Collection and analysis of data for diagnostic purposes

ABSTRACT

Systems and methods for determining bacterial load in targets and tracking changes in bacterial load of targets over time are disclosed. An autofluorescence detection and collection device includes a light source configured to directly illuminate at least a portion of a target and an area around the target with excitation light causing at least one biomarker in the illuminated target to fluoresce. Bacterial autofluorescence data regarding the illuminated portion of the target and the area around the target is collected and analyzed to determine bacterial load of the illuminated portion of the target and area around the target. The autofluorescence data may be analyzed using pixel intensity. Changes in bacterial load of the target over time may be tracked. The target may be a wound in tissue.

This application is a U.S. national stage application under 35 U.S.C. §371(c) of International Application No. PCT/CA2015/000444, filed Jul.24, 2015, which claims the benefit of priority to U.S. ProvisionalApplication No. 62/028,386, filed Jul. 24, 2014 (now expired), theentire content of each of which is incorporated by reference herein.

TECHNICAL FIELD

Devices and methods for collecting data for diagnostic purposes aredisclosed. In particular, the devices and methods of the presentapplication may be suitable for evaluating and tracking bacterial loadin a wound over time.

BACKGROUND

Wound care is a major clinical challenge. Healing and chronicnon-healing wounds are associated with a number of biological tissuechanges including inflammation, proliferation, remodeling of connectivetissues and, a common major concern, bacterial infection. A proportionof wound infections are not clinically apparent and contribute to thegrowing economic burden associated with wound care, especially in agingpopulations. Currently, the gold-standard wound assessment includesdirect visual inspection of the wound site under white light combinedwith indiscriminate collection of bacterial swabs and tissue biopsiesresulting in delayed, costly and often insensitive bacteriologicalresults. This may affect the timing and effectiveness of treatment.Qualitative and subjective visual assessment only provides a gross viewof the wound site, but does not provide information about underlyingbiological and molecular changes that are occurring at the tissue andcellular level. A relatively simple and complementary method thatcollects and analyzes ‘biological and molecular’ information inreal-time to provide early identification of such occult change andguidance regarding treatment of the same is desirable in clinical woundmanagement. Early recognition of high-risk wounds may guide therapeuticintervention and provide response monitoring over time, thus greatlyreducing both morbidity and mortality due especially to chronic wounds.

SUMMARY

In accordance with various exemplary embodiments, a method ofdetermining bacterial load of a target from fluorescent image data ofthe target is provided. The method comprises identifying a region ofinterest in a fluorescent image of a target, separating RGB images intoindividual channels, converting individual green and red image channelsfrom the RGB image to gray scale, and counting pixels whose gray scaleintensity was above a given threshold.

In accordance with another aspect of the present teachings, a method ofobtaining diagnostic data regarding a target is provided. The methodcomprises directly illuminating at least a portion of a target with ahomogeneous field of excitation light emitted by at least one lightsource connected to a housing of a handheld device, the housingincluding an enclosure for receiving a wireless communication devicehaving a digital camera. The at least one light source emits at leastone wavelength or wavelength band causing at least one biomarker in theilluminated portion of the target to fluoresce. The method furthercomprises collecting bacterial autofluorescence data regarding theilluminated portion of the target with an image sensor of the digitalcamera of the wireless communication device. The wireless communicationdevice is secured in the housing. The method also comprises analyzingthe collected bacterial autofluorescence data using pixel intensity todetermine bacterial load of the illuminated portion of the target.

In accordance with a further aspect of the present disclosure, a systemfor acquiring data regarding a wound in tissue is disclosed. The systemcomprises at least one light source configured to directly illuminate atarget surface with a homogeneous field of excitation light. The targetsurface includes at least a portion of a wound and an area around thewound. An optical sensor is configured to detect signals responsive toillumination of the illuminated portion of the wound and the area aroundthe wound. Each detected signal is indicative of at least one ofendogenous fluorescence, exogenous fluorescence, absorbance, andreflectance in the illuminated portion of the wound and the area aroundthe wound. A processor is configured to receive the detected signals andto analyze the detected signal data using pixel intensity and to outputdata regarding the bacterial load of the illuminated portion of thewound and area around the wound. The system further comprises a displayfor displaying the output data regarding the illuminated portion of thewound and the area around the wound output by the processor.

In accordance with yet another aspect of the present disclosure, aportable, handheld device for imaging and collection of data relating toa wound in tissue is disclosed. The device comprises a housingcomprising an enclosure configured to receive a mobile communicationdevice and at least one light source coupled to the housing andconfigured to directly illuminate at least a portion of a wound and anarea around the wound with a homogeneous field of light. A mobilecommunication device is secured in the enclosure of the housing, themobile communication device comprising an embedded digital camera andhaving a touchscreen display disposed on a first side of the device anda lens of the camera disposed on a second side of the device oppositethe first side. The mobile communication device is received in thehousing such that an image sensor of the digital camera is positioned todetect optical signals responsive to illumination of the portion of thewound and the area around the wound with the homogeneous field of light,each of the optical signals being indicative of at least one ofendogenous fluorescence, exogenous fluorescence, reflectance, andabsorbance in the illuminated portion of the wound and the area aroundthe wound. When the mobile communication device is secured in theenclosure, at least a portion of the touchscreen display is accessibleand viewable by a user. The device further comprises a processorconfigured to receive the detected optical signals, to analyze detectedsignal data using pixel intensity, and to output data regarding thebacterial load of the illuminated portion of the wound and area aroundthe wound.

In accordance with another aspect of the present disclosure, a method ofobtaining diagnostic data regarding a target is provided. The methodcomprises directly illuminating at least a portion of a target and anarea around the target with a homogeneous field of excitation lightemitted by at least one light source connected to a housing of ahandheld device. The housing includes an enclosure for receiving awireless communication device having a digital camera. The at least onelight source emits at least one wavelength or wavelength band causing atleast one biomarker in the illuminated portion of the target and areaaround the target to fluoresce. The method further comprises collectingbacterial autofluorescence data regarding the illuminated portion of thetarget and the area around the target with an image sensor of thedigital camera of the wireless communication device. The wirelesscommunication device is secured in the housing. The method furthercomprises analyzing the collected bacterial autofluorescence data todetermine bacterial load of the illuminated portion of the target andarea around the target, and tracking changes in bacterial load of thetarget over time.

Additional objects and advantages of the disclosure will be set forth inpart in the description which follows, and in part will be obvious fromthe description, or may be learned by practice of the disclosure. Theobjects and advantages of the disclosure will be realized and attainedby means of the elements and combinations particularly pointed out inthe appended claims.

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the disclosure, as claimed.

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate embodiments of the disclosure andtogether with the description, serve to explain the principles of thedisclosure.

BRIEF DESCRIPTION OF DRAWINGS

At least some features and advantages will be apparent from thefollowing detailed description of embodiments consistent therewith,which description should be considered with reference to theaccompanying drawings, wherein:

FIG. 1 is a schematic diagram of a device for fluorescence-basedmonitoring;

FIG. 2 shows an example of a clinical wound care facility using a devicefor fluorescence-based monitoring;

FIG. 3 shows images of a muscle surface of a pig meat sample,demonstrating the use of a device for fluorescence-based monitoring forautofluorescence detection of connective tissues and bacteria;

FIG. 4 shows images of a hand-held embodiment of a device forfluorescence-based monitoring;

FIG. 5 shows an alternate embodiment of a handheld device for obtainingwhite light and fluorescent light data from a target;

FIGS. 6A and 6B show another alternative embodiment of a handheld devicefor obtaining data regarding a target, the handheld device incorporatingan iPhone;

FIGS. 7A and 7B illustrate exemplary methods of determining bacterialload of a target;

FIG. 8 shows representative white light (WL) and fluorescent (FL) imagesfor a single mouse tracked over 10 days;

FIG. 9 illustrates preclinical data which show that pathogenic bacterialautofluorescence (AF) intensity correlates with bacterial load in vivo;

FIG. 10 shows images of live bacterial cultures captured using a devicefor fluorescence-based monitoring;

FIG. 11 shows an example of bacterial monitoring using a device forfluorescence-based monitoring;

FIG. 12 shows images of a simulated animal wound model, demonstratingnon-invasive autofluorescence detection of bacteria using a device forfluorescence-based monitoring;

FIG. 13 illustrates an example of monitoring of a chronic wound;

FIGS. 14-28 show examples of the use of a device for fluorescence-basedmonitoring for imaging wounds and conditions in clinical patients;

FIG. 29 shows images of a skin surface of a pig meat sample,demonstrating non-invasive autofluorescence detection of collagen andvarious bacterial species using a device for fluorescence-basedmonitoring;

FIG. 30 shows images and spectral plots demonstrating the use of adevice for fluorescence-based monitoring to detect fluorescence frombacteria growing in agar plates and on the surface a simulated wound onpig meat;

FIG. 31 shows images demonstrating use of a device forfluorescence-based monitoring for imaging of blood and microvasculature;

FIG. 32 is a flowchart illustrating the management of a chronic woundusing a device for fluorescence-based monitoring;

FIG. 33 illustrates the phases of wound healing with time;

FIG. 34 is a table showing examples of tissue, cellular and molecularbiomarkers known to be associated with wound healing;

FIG. 35 is a diagram comparing a healthy wound to a chronic wound;

FIG. 36 shows images demonstrating the use of a device forfluorescence-based monitoring in imaging a mouse model; and

FIG. 37 shows an example of the use of a device for fluorescence-basedmonitoring for imaging small animal models;

FIG. 38 shows an example of a kit including a device forfluorescence-based monitoring.

Although the following detailed description makes reference toillustrative embodiments, many alternatives, modifications, andvariations thereof will be apparent to those skilled in the art.Accordingly, it is intended that the claimed subject matter be viewedbroadly.

DETAILED DESCRIPTION

Reference will now be made in detail to various embodiments, examples ofwhich are illustrated in the accompanying drawings. The variousexemplary embodiments are not intended to limit the disclosure. To thecontrary, the disclosure is intended to cover alternatives,modifications, and equivalents.

Conventional clinical assessment methods of acute and chronic woundscontinue to be suboptimal. Such assessment methods usually are based ona complete patient history, qualitative and subjective clinicalassessment with simple visual appraisal using ambient white light andthe ‘naked eye,’ and can sometimes involve the use of color photographyto capture the general appearance of a wound under white lightillumination. Regular re-assessment of progress toward healing andappropriate modification of the intervention is also necessary. Woundassessment terminology is non-uniform, many questions surrounding woundassessment remain unanswered, agreement has yet to be reached on the keywound parameters to measure in clinical practice, and the accuracy andreliability of available wound assessment techniques vary.

Visual assessment is frequently combined with swabbing and/or tissuebiopsies for bacteriological culture for diagnosis. Bacterial swabs arecollected at the time of wound examination and have the noted advantageof providing identification of specific bacterial/microbial species.However, multiple swabs and/or biopsies often are collected randomlyfrom the wound site, and some swabbing techniques may in fact spread themicroorganisms around with the wound during the collection process thusaffecting patient healing time and morbidity. This may be a problemespecially with large chronic (non-healing) wounds where the detectionyield for bacterial presence using current swabbing and biopsy protocolsis suboptimal (diagnostically insensitive), despite many swabs beingcollected.

Thus, current methods for obtaining swabs or tissue biopsies from thewound site for subsequent bacteriological culture are based on anon-targeted or ‘blind’ swabbing or punch biopsy approach, and have notbeen optimized to minimize trauma to the wound or to maximize thediagnostic yield of the bacteriology tests. In addition, bacteriologicalculture results often take about 2-3 days to come back from thelaboratory and can be inconclusive, thus delaying accurate diagnosis andtreatment. Thus, conventional methods of obtaining bacterial swabs donot necessarily provide relevant data regarding the wound and cannotprovide real-time detection of infectious status of wounds. The lack ofa non-invasive method to objectively and rapidly evaluate wound repairat a biological level (which may be at greater detail than simplyappearance or morphology based), and to aid in targeting of thecollection of swab and tissue biopsy samples for bacteriology is a majorobstacle in clinical wound assessment and treatment. An alternativemethod is highly desirable.

As wounds (chronic and acute) heal, a number of key biological changesoccur at the wound site at the tissue and cellular level. Wound healinginvolves a complex and dynamic interaction of biological processesdivided into four overlapping phases—haemostasis, inflammation, cellularproliferation, and maturation or remodeling of connective tissues—whichaffect the pathophysiology of wound healing. A common major complicationarising during the wound healing process, which can range from days tomonths, is infection caused by bacteria and other microorganisms. Thiscan result in a serious impediment to the healing process and lead tosignificant complications. All wounds contain bacteria at levels rangingfrom contamination, through colonization, critical colonization toinfection, and diagnosis of bacterial infection is based on clinicalsymptoms and signs (e.g., visual and odorous cues).

The most commonly used terms for wound infection have included woundcontamination, wound colonisation, wound infection and, more recently,critical colonisation. Wound contamination refers to the presence ofbacteria within a wound without any host reaction; wound colonisationrefers to the presence of bacteria within the wound which do multiply orinitiate a host reaction; and critical colonisation refers tomultiplication of bacteria causing a delay in wound healing, usuallyassociated with an exacerbation of pain not previously reported butstill with no overt host reaction. Wound infection refers to thedeposition and multiplication of bacteria in tissue with an associatedhost reaction. In practice the term ‘critical colonisation’ can be usedto describe wounds that are considered to be moving from colonisation tolocal infection. The challenge within the clinical setting, however, isto ensure that this situation is quickly recognized with confidence andfor the bacterial bioburden to be reduced as soon as possible, perhapsthrough the use of topical antimicrobials. Potential wound pathogens canbe categorised into different groups, such as, bacteria, fungi, spores,protozoa and viruses depending on their structure and metaboliccapabilities. Although viruses do not generally cause wound infections,bacteria can infect skin lesions formed during the course of certainviral diseases. Such infections can occur in several settings includingin health-care settings (hospitals, clinics) and at home or chronic carefacilities. The control of wound infections is increasingly complicated,yet treatment is not always guided by microbiological diagnosis. Thediversity of micro-organisms and the high incidence of polymicrobicflora in most chronic and acute wounds give credence to the value ofidentifying one or more bacterial pathogens from wound cultures. Theearly recognition of causative agents of wound infections can assistwound care practitioners in taking appropriate measures. Furthermore,faulty collagen formation arises from increased bacterial burden andresults in over-vascularized friable loose granulation tissue thatusually leads to wound breakdown.

Accurate and clinically relevant wound assessment is an importantclinical tool, but this process currently remains a substantialchallenge. Current visual assessment in clinical practice only providesa gross view of the wound site (e.g., presence of purulent material andcrusting). Current best clinical practice fails to adequately use thecritically important objective information about underlying keybiological changes that are occurring at the tissue and cellular level(e.g., contamination, colonization, infection, matrix remodeling,inflammation, bacterial/microbial infection, and necrosis) since suchindices are i) not easily available at the time of the wound examinationand ii) they are not currently integrated into the conventional woundmanagement process. Direct visual assessment of wound health statususing white light relies on detection of color andtopographical/textural changes in and around the wound, and thus may beincapable and unreliable in detecting subtle changes in tissueremodeling. More importantly, direct visual assessment of wounds oftenfails to detect the presence of bacterial infection, since bacteria areoccult under white light illumination. Infection is diagnosed clinicallywith microbiological tests used to identify organisms and theirantibiotic susceptibility. Although the physical indications ofbacterial infection can be readily observed in most wounds using whitelight (e.g., purulent exudate, crusting, swelling, erythema), this isoften significantly delayed and the patient is already at increased riskof morbidity (and other complications associated with infection) andmortality. Therefore, standard white light direct visualization fails todetect the early presence of the bacteria themselves or identify thetypes of bacteria within the wound.

Wound progression is currently monitored manually. The National PressureUlcer Advisory Panel (NPUAP) developed the Pressure Ulcer Scale forHealing (PUSH) tool that outlines a five-step method of characterizingpressure ulcers. This tool uses three parameters to determine aquantitative score that is then used to monitor the pressure ulcer overtime. The qualitative parameters include wound dimensions, tissue type,and the amount of exudate or discharge, and thermal readings presentafter the dressing is removed. A wound can be further characterized byits odor and color. Such an assessment of wounds currently does notinclude critical biological and molecular information about the wound.Therefore, all descriptions of wounds are somewhat subjective and notedby hand by either the attending physician or the nurse.

What is desirable is a robust, cost-effective non-invasive and rapidimaging-based method or device for collecting wound data and providingan analysis in real-time. The data and analysis can be used toobjectively assess wounds for changes at the biological, biochemical andcellular levels and to rapidly, sensitively and non-invasively detectingthe earliest presence of bacteria/microorganisms within wounds. Such amethod or device for detection of critical biological tissue changes inwounds may serve an adjunctive role with conventional clinical woundmanagement methods in order to guide key clinico-pathological decisionsin patient care. Such a device may be compact, portable and capable ofreal-time non-invasive and/or non-contact interrogation of wounds in asafe and convenient manner, which may allow it to fit seamlessly intoroutine wound management practice and user friendly to the clinician,nurse and wound specialist. This may also include use of this device inthe home-care environment (including self-use by a patient), as well asin military battlefield environments. In addition, such an image-baseddevice may provide an ability to monitor wound treatment response andhealing in real-time by incorporating valuable ‘biologically-informed’image-guidance into the clinical wound assessment process. This mayultimately lead to potential new diagnosis, treatment planning,treatment response monitoring and thus ‘adaptive’ interventionstrategies which may permit enhancement of wound-healing response at theindividual patient level. Precise identification of the systemic, local,and molecular factors underlying the wound healing problem in individualpatients may allow better tailored treatment.

In accordance with the present teachings, methods of analysis for datacollected from a wound are provided. For example, the collection offluorescence image data appears to be promising for improving clinicalwound assessment and management. When excited by short wavelength light(e.g., ultraviolet or short visible wavelengths), most endogenousbiological components of tissues (e.g., connective tissues such collagenand elastins, metabolic co-enzymes, proteins, etc.) produce fluorescenceof a longer wavelength, in the ultraviolet, visible, near-infrared andinfrared wavelength ranges.

Tissue autofluorescence imaging provides a unique means of obtainingbiologically relevant information of normal and diseased tissues inreal-time, thus allowing differentiation between normal and diseasedtissue states. This is based, in part, on the inherently differentlight-tissue interactions (e.g., absorption and scattering of light)that occur at the bulk tissue and cellular levels, changes in the tissuemorphology and alterations in the blood content of the tissues. Intissues, blood is a major light absorbing tissue component (i.e., achromophore). This type of technology is suited for imaging disease inhollow organs (e.g., GI tract, oral cavity, lungs, bladder) or exposedtissue surfaces (e.g., skin). An autofluorescence imaging device inaccordance with the presnet teachings may collect wound data thatprovides/allows rapid, non-invasive and non-contact real-time analysisof wounds and their composition and components, to detect and exploitthe rich biological information of the wound to improve clinical careand management.

A device in accordance with the present disclosure 1) providesimage-guidance for tissue sampling, detecting clinically-significantlevels of pathogenic bacteria and wound infection otherwise overlookedby conventional sampling and 2) provides image-guidance for woundtreatment, accelerating wound closure compared with conventionaltherapies and quantitatively tracking long-term changes in bacterialbioburden and distribution in wounds.

U.S. Pat. No. 9,042,967 B2 to DaCosta et al., entitled “Device andMethod for Wound Imaging and Monitoring,” and issued on May 26, 2015,discloses at least some aspects of a device configured to collect datafor objectively assessing wounds for changes at the biological,biochemical and cellular levels and for rapidly, sensitively andnon-invasively detecting the earliest presence ofbacteria/microorganisms within wounds. This patent claims priority toPCT Application No. PCT/CA2009/000680 filed on May 20, 2009, and to U.S.Provisional Patent Application No. 61/054,780, filed on May 20, 2008.The entire content of each of these above-identified patents, patentapplications, and patent application publications is incorporated hereinby reference.

In accordance with one aspect of the present teachings, a handheldportable device to examine skin and wounds in real-time is provided. Thedevice instantly detects, visualizes, and analyzes bacteria and tissuecomposition. The device is a compact, hand-held, device for noncontactand noninvasive imaging. It captures both white light (WL) andautofluorescence (AF) signals produced by tissue components and bacteriawithout the use of contrast agents. Although capable of detecting AFsignals without use of contrast agents, one of ordinary skill in the artwill understand that the devices disclosed herein can be used withcontrast agents if desired. In addition to white light and fluorescence,the device also may capture thermal data from the imaged area. Thedevice may be further configured to analyze the white light,fluorescence, and thermal data, correlate such data, and provide anoutput based on the correlation of the data, such as, for example, anindication of wound status, wound healing, wound infection, bacterialload, or other diagnostic information upon which an interventionstrategy may be based.

The device may be configured to create and/or display composite imagesincluding green AF, produced by endogenous connective tissues (e.g.,collagen, elastin) in skin, and red AF, produced by endogenousporphyrins in clinically relevant bacteria such as Staphylococcusaureus. Siderophores/pyoverdins in other species such as Pseudomonasaeruginosa appear blue-green in color with in vivo AF imaging. Thedevice may provide visualization of bacterial presence, types,distribution, amounts in and around a wound as well as key informationsurrounding tissue composition (collagen, tissue viability, blood oxygensaturation). For example, the device may provide imaging of collagencomposition in and around skin in real-time (via AF imaging).

In accordance with the present disclosure, the device may be configuredto accurately detect and measure bacterial load in wounds in real-time,guide treatment decisions, and track wound healing over the course ofantibacterial treatment. Additionally, bioluminescence imaging (BLI) maybe used to correlate absolute bacterial load with FL signals obtainedusing the handheld device.

The device may be independent and self-contained. It may interface withcomputers, printers and EMR systems.

In accordance with one exemplary embodiment of the present disclosure,the device is configured to image bacteria in real-time (via, forexample, fluorescence imaging), permitting ready identification ofbacteria types, their location, distribution and quantity in acceptedunits of measurement and allowing identification of and distinctionbetween several different species of bacteria. For example,autofluorescence imaging may be used to visualize and differentiatePseudomonas aruginosa (which fluoresces a greenish-blue colour whenexcited by 405 nm light from the device) from other bacteria (e.g.,Staphylococcus aureus) that predominantly fluoresce a red/orange colourunder the same excitation wavelength. In one exemplary embodiment thedevice's camera sensor and built in fluorescence multiband pass emissionfilter produce fluorescence images of bacteria (in wounds or normalskin) and Pseudomonas aruginosa appear greenish-blue in colour whileother bacteria emit a red/orange colour. The device detects differencesin the autofluorescence emission of different endogenous molecules(called fluorophores) between the different bacteria.

In accordance with another exemplary embodiment of the presentdisclosure, the device is configured to identify or provide anindication of tissue viability in real-time (via fluorescence imaging).For example, blood preferentially absorbs 405 nm light compared withother visible wavelengths. Tissues which are perfused by blood areconsidered viable, and can be differentiated from devitalized (poorlyperfused) tissues using fluorescence imaging. Using 405 nm light from adevice in accordance with the present teachings to illuminate a wound,the device can be configured with a multiband pass emission filter todetect the amount of 405 nm light that is absorbed or reflected from thetissues. Viable tissue contains blood that highly absorbs 405 nm lightresulting in an image with low levels of 405 nm light, whereas nonviable(or devitalized) tissues do not contain sufficient blood and 405 nm isless absorbed. Thus, in an image of a wound where viable and nonviabletissues are present, the user will recognize viable tissues (fromnonviable tissues) based on the relative amount of 405 nm light in theimage, the viable tissues appearing darker compared with the nonviabletissues. In addition, in the green fluorescence “channel” of theresultant image (of the wound), viable tissues will appear less greenfluorescent compared with nonviable tissues because viable tissues willpreferentially absorb more of the 405 nm excitation light due to moreblood being present, compared with nonviable tissues. Thus, while bothviable and nonviable tissues in a resultant image obtained by the devicemay contain similar amounts of green fluorescent connective tissues(i.e., collagens), viable tissue will have less 405 nm excitation lightto stimulate the connective tissue autofluorescence than nonviabletissues. The result is that viable tissues will have less greenconnective tissue fluorescence than non-viable tissues in the sameimage. The user will appreciate this difference visually during imagingwith the device.

In accordance with another aspect of the present disclosure, the deviceis configured to capture and generate images and videos that provide amap or other visual display of user selected parameters. Such maps ordisplays may correlate, overlay, co-register or otherwise coordinatedata generated by the device based on input from one or more devicesensors. Such sensors may include, for example, camera sensorsconfigured to detect white light and/or fluorescent images and thermalsensors configured to detect heat signatures of a target. For example,the device may be configured to display color images, image maps, orother maps of user selected parameters such as, for example, bacterialocation and/or biodistribution, collagen location, location anddifferentiation between live tissues and dead tissues, differentiationbetween bacterial species, location and extent of blood, bone, exudate,temperature and wound area/size. These maps or displays may be output bythe device based on the received signals and may be produced on a singleimage with or without quantification displays. The user-selectedparameters shown on the map may be correlated with one or more woundparameters, such as shape, size, topography, volume, depth, and area ofthe wound. For example, in accordance with one exemplary embodiment, itis possible to use a ‘pseudo-coloured’ display of the fluorescenceimages/videos of wounds to color-code bacteria fluorescence (one colour)and connective tissues (another colour) etc. This may be accomplishedby, for example, using a pixel-by-pixel coloring based on the relativeamount of 405 nm light in the Blue channel of the resultant RGB image,green connective tissue fluorescence in the Green channel, and redbacteria fluorescence in Red channel. Additionally and/or alternatively,this may be accomplished by displaying the number of pixels in a givenimage for each of the blue, green and red channels which would representamount of blood in tissue, amount of connective tissues, and amount ofbacteria, respectively.

In accordance with one aspect of the present disclosure, the device maybe configured to create and output reports regarding the collected data.For example, in accordance with one exemplary embodiment, the deviceuser can generate a wound status report, which may include, for example,date/time, patient ID, images, etc. The user can export or print images,to a selected network, computer, printer when connected to cradle,and/or via USB to computer. The reports may be generated by the handhelddevice, by exporting data to a computer for processing and generation ofreports, or by a combination of the two. Further, such reports, or thedata contained therein, may form the basis of recommended interventionor treatment strategies. Reports may include, for example, medicalreports, digital reports, reports that encompass handwritten input fromclinicians (e.g., via tablet input, etc.). The reports may includevarious types of data including, for example, the identification ofwound parameters and the tracking of these parameters over time. Forexample, the reports may identify and track changes in wound size, woundshape, wound topography, wound volume, wound area, bacterial load of thewound, location of bacteria within the wound, presence of exposed bone,blood, connective and other tissues, wound temperature, location of thewound on the patient, number of wounds on the patient, date of woundexamination, patient identification, medications administered to thepatient, interventional strategies and therapies as administered and aschanged over time in response to changing wound parameters, etc. Forexample, the device may generate a report that tracks a patient's woundand skin status changes, including for example, wound size and bacterialburden over time. Further, the data collected may be used to generate adatabase that provides clinical data regarding wound parameters and theefficacy of various wound intervention/treatment strategies.Additionally, the device may be configured to integrate collecteddata/images/videos into the reports and, alternatively or additionally,include such reports and data/images/videos into a patient's electronicmedical record (EMR). This process may be wirelessly, via the use oftransfer cables, and the system also may be configured to upload thereports automatically.

The device has a memory sufficient to store several images/videos. Inaddition to internal memory, the device may include a Micro SD cardinterface for additional storage and firmware development. The devicecan inform the user of low memory capacity. The device may also includea data safeguard that will prompt a user to export files in the case oflow memory availability.

In accordance with one aspect of the present disclosure, a method anddevice for fluorescence-based imaging and monitoring is disclosed. Oneexemplary embodiment of the device is a portable optical digital imagingdevice. The device may utilize a combination of white light, tissuefluorescence and reflectance imaging, and thermal imaging, and mayprovide real-time wound imaging, assessment, recordation/documentation,monitoring and/or care management. The device may be hand-held, compactand/or light-weight. This device and method may be suitable formonitoring of wounds in humans and animals.

The device may generally comprise: i) one or moreexcitation/illumination light sources and ii) a detector device (e.g., adigital imaging detector device), which may be combined with one or moreoptical emission filters, or spectral filtering mechanisms, and whichmay have a view/control screen (e.g., a touch-sensitive screen), imagecapture and zoom controls. The device may also have: iii) a wired and/orwireless data transfer port/module, iv) an electrical power source andpower/control switches, and/or v) an enclosure, which may be compactand/or light weight, and which may have a mechanism for attachment ofthe detector device and/or a handle grip. The excitation/illuminationlight sources may be LED arrays emitting light at about 405 nm (e.g.,+/−5 nm), and may be coupled with additional band-pass filters centeredat about 405 nm to remove/minimize the side spectral bands of light fromthe LED array output so as not to cause light leakage into the imagingdetector with its own optical filters. The digital imaging detectordevice may be a digital camera, for example having at least an ISO800sensitivity, but more preferably an ISO3200 sensitivity, and may becombined with one or more optical emission filters, or other equallyeffective (e.g., miniaturized) mechanized spectral filtering mechanisms(e.g., acousto-optical tunable filter or liquid crystal tunable filter).The digital imaging detector device may have a touch-sensitive viewingand/or control screen, image capture and zoom controls. The enclosuremay be an outer hard plastic or polymer shell, enclosing the digitalimaging detector device, with buttons such that all necessary devicecontrols may be accessed easily and manipulated by the user. Miniatureheat sinks or small mechanical fans, or other heat dissipating devicesmay be embedded in the device to allow excess heat to be removed fromthe excitation light sources if required. The complete device, includingall its embedded accessories and attachments, may be powered usingstandard AC/DC power and/or by rechargeable battery pack. The completedevice may also be attached or mounted to an external mechanicalapparatus (e.g., tripod, or movable stand with pivoting arm) allowingmobility of the device within a clinical room with hands-free operationof the device. Alternatively, the device may be provided with a mobileframe such that it is portable. The device may be cleaned using moistgauze wet with water, while the handle may be cleansed with moist gauzewet with alcohol. Additional appropriate cleaning methods will beapparent to those of ordinary skill in the art. The device may includesoftware allowing a user to control the device, including control ofimaging parameters, visualization of images, storage of image data anduser information, transfer of images and/or associated data, and/orrelevant image analysis (e.g., diagnostic algorithms).

A schematic diagram of an example of the device is shown in FIG. 1. Thedevice is shown positioned to image a target object 10 or targetsurface. In the example shown, the device has a digital imageacquisition device 1, such as digital camera, video recorder, camcorder,cellular telephone with built-in digital camera, ‘Smart’ phone with adigital camera, personal digital assistant (PDA), laptop/PC with adigital camera, or a webcam. The digital image acquisition device 1 hasa lens 2, which may be aligned to point at the target object 10 and maydetect the optical signal that emanates from the object 10 or surface.The device has an optical filter holder 3 which may accommodate one ormore optical filters 4. Each optical filter 4 may have differentdiscrete spectral bandwidths and may be band-pass filters. These opticalfilters 4 may be selected and moved in from of the digital camera lensto selectively detect specific optical signals based on the wavelengthof light. The device may include light sources 5 that produce excitationlight to illuminate the object 10 in order to elicit an optical signal(e.g., fluorescence) to be imaged with, for example, blue light (e.g.,400-450 nm), or any other combination of single or multiple wavelengths(e.g., wavelengths in the ultraviolet/visible/near infrared/infraredranges). The light source 5 may comprise a LED array, laser diode and/orfiltered lights arranged in a variety of geometries. The device mayinclude a method or apparatus 6 (e.g., a heatsink or a cooling fan) todissipate heat and cool the illumination light sources 5. The device mayinclude a method or apparatus 7 (e.g., an optical band-pass filter) toremove any undesirable wavelengths of light from the light sources 5used to illuminate the object 10 being imaged. The device may include amethod or apparatus 8 to use an optical means (e.g., use of compactminiature laser diodes that emit a collimated light beam) to measure anddetermine the distance between the imaging device and the object 10. Forexample, the device may use two light sources, such as two laser diodes,as part of a triangulation apparatus to maintain a constant distancebetween the device and the object 10. Other light sources may bepossible. The device may also use ultrasound, or a physical measure,such as a ruler, to determine a constant distance to maintain. Inaccordance with another exemplary embodiment, the device may use arangefinder to determine the appropriate position of the device relativeto the wound to be imaged. The device may also include a method orapparatus 9 (e.g., a pivot) to permit the manipulation and orientationof the excitation light sources 5, 8 so as to manoeuvre these sources 5,8 to change the illumination angle of the light striking the object 10for varying distances.

The target object 10 may be marked with a mark 11 to allow for multipleimages to be taken of the object and then being co-registered foranalysis. The mark 11 may involve, for example, the use of exogenousfluorescence dyes of different colours which may produce multipledistinct optical signals when illuminated by the light sources 5 and bedetectable within the image of the object 10 and thus may permitorientation of multiple images (e.g., taken over time) of the sameregion of interest by co-registering the different colours and thedistances between them. The digital image acquisition device 1 mayinclude one or more of: an interface 12 for a head-mounted display; aninterface 13 for an external printer; an interface 14 for a tabletcomputer, laptop computer, desk top computer or other computer device;an interface 15 for the device to permit wired or wireless transfer ofimaging data to a remote site or another device; an interface 16 for aglobal positioning system (GPS) device; an interface 17 for a deviceallowing the use of extra memory; and an interface 18 for a microphone.

The device may include a power supply 19 such as an AC/DC power supply,a compact battery bank, or a rechargeable battery pack. Alternatively,the device may be adapted for connecting to an external power supply.The device may have a housing 20 that houses all the components in oneentity. The housing 20 may be equipped with a means of securing anydigital imaging device within it. The housing 20 may be designed to behand-held, compact, and/or portable. The housing 20 may be one or moreenclosures.

FIG. 2 shows an example of the device in a typical wound care facility.Inset a) shows a typical clinical wound care facility, showing theexamination chair and accessory table. Insets b-c) show an example ofthe device in its hard-case container. The device may be integrated intothe routine wound care practice allowing real-time imaging of thepatient. Inset d) shows an example of the device (arrow) placed on the“wound care cart” to illustrate the size of the device. Inset e) Thedevice may be used to image under white light illumination, while insetf) shows the device being used to take fluorescence images of a woundunder dimmed room lights. Inset g) the device may be used intelemedicine/telehealth infrastructures, for example fluorescence imagesof a patient's wounds may be sent by email to a wound care specialistvia a wireless communication device, such as a Smartphone at anotherhospital using a wireless/WiFi internet connection. Using this device,high-resolution fluorescence images may be sent as email attachments towound care specialists from remote wound care sites for immediateconsultation with clinical experts, microbiologists, etc. at specializedclinical wound care and management centers.

Examples

An example of a device for fluorescence-based monitoring is describedbelow. All examples are provided for the purpose of illustration onlyand are not intended to be limiting. Parameters such as wavelengths,dimensions, and incubation time described in the examples may beapproximate and are provided as examples only.

In this example, the devices uses two violet/blue light (e.g., 405nm+/−10 nm emission, narrow emission spectrum) LED arrays (Opto DiodeCorporation, Newbury Park, Calif.), each situated on either side of theimaging detector assembly as the excitation or illumination lightsources. These arrays have an output power of approximately 1 Watt each,emanating from a 2.5×2.5 cm², with a 70-degree illuminating beam angle.The LED arrays may be used to illuminate the tissue surface from adistance of about 10 cm, which means that the total optical powerdensity on the skin surface is about 0.08 W/cm². At such low powers,there is no known potential harm to either the target wound or skinsurface, or the eyes from the excitation light. However, it may beinadvisable to point the light directly at any individual's eyes duringimaging procedures. It should also be noted that 405 nm light does notpose a risk to health according to international standards formulated bythe International Electrotechnical Commission (IEC), as further detailedon the website:

http://www.iec.ch/online_news/etech/arch_2006/etech_0906/focus.htm

The one or more light sources may be articulated (e.g., manually) tovary the illumination angle and spot size on the imaged surface, forexample by using a built in pivot, and are powered for example throughan electrical connection to a wall outlet and/or a separate portablerechargeable battery pack. Excitation/illumination light may be producedby sources including, but not limited to, individual or multiplelight-emitting diodes (LEDs) in any arrangement including in ring orarray formats, wavelength-filtered light bulbs, or lasers. Selectedsingle and multiple excitation/illumination light sources with specificwavelength characteristics in the ultraviolet (UV), visible (VIS),far-red, near infrared (NIR) and infrared (IR) ranges may also be used,and may be composed of a LED array, organic LED, laser diode, orfiltered lights arranged in a variety of geometries.Excitation/illumination light sources may be ‘tuned’ to allow the lightintensity emanating from the device to be adjusted while imaging. Thelight intensity may be variable. The LED arrays may be attached toindividual cooling fans or heat sinks to dissipate heat produced duringtheir operation. The LED arrays may emit narrow 405 nm light, which maybe spectrally filtered using a commercially available band-pass filter(Chroma Technology Corp, Rockingham, Vt., USA) to reduce potential‘leakage’ of emitted light into the detector optics. When the device isheld above a tissue surface (e.g., a wound) to be imaged, theilluminating light sources may shine a narrow-bandwidth orbroad-bandwidth violet/blue wavelength or other wavelength or wavelengthband of light onto the tissue/wound surface thereby producing a flat andhomogeneous field within the region-of-interest. The light may alsoilluminate or excite the tissue down to a certain shallow depth. Thisexcitation/illumination light interacts with the normal and diseasedtissues and may cause an optical signal (e.g., absorption, fluorescenceand/or reflectance) to be generated within the tissue.

By changing the excitation and emission wavelengths accordingly, theimaging device may interrogate tissue components (e.g., connectivetissues and bacteria in a wound) at the surface and at certain depthswithin the tissue (e.g., a wound). For example, by changing fromviolet/blue (˜400-500 nm) to green (˜500-540 nm) wavelength light,excitation of deeper tissue/bacterial fluorescent sources may beachieved, for example in a wound. Similarly, by detecting longerwavelengths, fluorescence emission from tissue and/or bacterial sourcesdeeper in the tissue may be detected at the tissue surface. For woundassessment, the ability to interrogate surface and/or sub-surfacefluorescence may be useful, for example in detection and potentialidentification of bacterial contamination, colonization, criticalcolonization and/or infection, which may occur at the surface and oftenat depth within a wound (e.g., in chronic non-healing wounds). In oneexample, referring to FIG. 3, inset c) shows the detection of bacteriabelow the skin surface (i.e., at depth) after wound cleaning. This useof the device for detecting bacteria at the surface and at depth withina wound and surrounding tissue may be assessed in the context of otherclinical signs and symptoms used conventionally in wound care centers.

Example embodiments of the device are shown in FIG. 4. The device may beused with any standard compact digital imaging device (e.g., acharge-coupled device (CCD) or complementary metal-oxide-semiconductor(CMOS) sensors) as the image acquisition device. The example deviceshown in a) has an external electrical power source, the two LED arraysfor illuminating the object/surface to be imaged, and a commerciallyavailable digital camera securely fixed to light-weight metal frameequipped with a convenient handle for imaging. A multi-band filter isheld in front of the digital camera to allow wavelength filtering of thedetected optical signal emanating from the object/surface being imaged.The camera's video/USB output cables allow transfer of imaging data to acomputer for storage and subsequent analysis. This example uses acommercially-available 8.1-megapixel Sony digital camera (Sony CybershotDSC-T200 Digital Camera, Sony Corporation, North America). This cameramay be suitable because of i) its slim vertical design which may beeasily integrated into the enclosure frame, ii) its large 3.5-inchwidescreen touch-panel LCD for ease of control, iii) its Carl Zeiss 5×optical zoom lens, and iv) its use in low light (e.g., ISO 3200). Thedevice may have a built-in flash which allows for standard white lightimaging (e.g., high-definition still or video with sound recordingoutput). Camera interface ports may support both wired (e.g., USB) orwireless (e.g., Bluetooth, WiFi, and similar modalities) data transferor 3^(rd) party add-on modules to a variety of external devices, suchas: a head-mounted display, an external printer, a tablet computer,laptop computer, personal desk top computer, a wireless device to permittransfer of imaging data to a remote site/other device, a globalpositioning system (GPS) device, a device allowing the use of extramemory, and a microphone. The digital camera is powered by rechargeablebatteries, or AC/DC powered supply. The digital imaging device mayinclude, but is not limited to, digital cameras, webcams, digital SLRcameras, camcorders/video recorders, cellular telephones with embeddeddigital cameras, Smartphones™, personal digital assistants (PDAs), andlaptop computers/tablet PCs, or personal desk-top computers, all ofwhich contain/or are connected to a digital imaging detector/sensor.

This light signal produced by the excitation/illumination light sourcesmay be detected by the imaging device using optical filter(s) (e.g.,those available from Chroma Technology Corp, Rockingham, Vt., USA) thatreject the excitation light but allow selected wavelengths of emittedlight from the tissue to be detected, thus forming an image on thedisplay. There is an optical filter holder attached to the enclosureframe in from of the digital camera lens which may accommodate one ormore optical filters with different discrete spectral bandwidths, asshown in insets b) and c) of FIG. 4. Inset b) shows the device with theLED arrays turned on to emit bright violet/blue light, with a singleemission filter in place. Inset c) shows the device using amultiple-optical filter holder used to select the appropriate filter fordesired wavelength-specific imaging. Inset d) shows the device beingheld in one hand while imaging the skin surface of a foot.

These band-pass filters may be selected and aligned in front of thedigital camera lens to selectively detect specific optical signals fromthe tissue/wound surface based on the wavelength of light desired.Spectral filtering of the detected optical signal (e.g., absorption,fluorescence, reflectance) may also be achieved, for example, using aliquid crystal tunable filter (LCTF), or an acousto-optic tunable filter(AOTF) which is a solid-state electronically tunable spectral band-passfilter. Spectral filtering may also involve the use of continuousvariable filters, and/or manual band-pass optical filters. These devicesmay be placed in front of the imaging detector to produce multispectral,hyperspectral, and/or wavelength-selective imaging of tissues.

The device may be modified by using optical or variably orientedpolarization filters (e.g., linear or circular combined with the use ofoptical wave plates) attached in a reasonable manner to theexcitation/illumination light sources and the imaging detector device.In this way, the device may be used to image the tissue surface withpolarized light illumination and non-polarized light detection or viceversa, or polarized light illumination and polarized light detection,with either white light reflectance and/or fluorescence imaging. Thismay permit imaging of wounds with minimized specular reflections (e.g.,glare from white light imaging), as well as enable imaging offluorescence polarization and/or anisotropy-dependent changes inconnective tissues (e.g., collagens and elastin) within the wound andsurrounding normal tissues. This may yield useful information about thespatial orientation and organization of connective tissue fibersassociated with wound remodeling during healing.

All components of the imaging device may be integrated into a singlestructure, such as an ergonomically designed enclosed structure with ahandle, allowing it to be comfortably held with one or both hands. Thedevice may also be provided without any handle. The device may be lightweight, portable, and may enable real-time digital imaging (e.g., stilland/or video) of any target surface (for example, the skin and/or oralcavity, which is also accessible) using white light, fluorescence and/orreflectance imaging modes. The device may be scanned across the bodysurface for imaging by holding it at variable distances from thesurface, and may be used in a lit environment/room to image white lightreflectance/fluorescence. The device may be used in a dim or darkenvironment/room to optimize the tissue fluorescence signals, andminimize background signals from room lights. The device may be used fordirect (e.g., with the unaided eye) or indirect (e.g., via the viewingscreen of the digital imaging device) visualization of wounds andsurrounding normal tissues.

The device may also be embodied as not being hand-held or portable, forexample as being attached to a mounting mechanism (e.g., a tripod orstand) for use as a relatively stationary optical imaging device forwhite light, fluorescence and reflectance imaging of objects, materials,and surfaces (e.g., a body). This may allow the device to be used on adesk or table or for ‘assembly line’ imaging of objects, materials andsurfaces. In some embodiments, the mounting mechanism may be mobile.

Other features of this device may include the capability of digitalimage and video recording, possibly with audio, methods fordocumentation (e.g., with image storage and analysis software), andwired or wireless data transmission for remote telemedicine/E-healthneeds. For example, insets e) and f) of FIG. 4 show an embodiment of thedevice where the image acquisition device is a mobile communicationdevice such as a cellular telephone. The cellular telephone used in thisexample is a Samsung Model A-900, which is equipped with a 1.3 megapixeldigital camera. The telephone is fitted into the holding frame forconvenient imaging. Inset e) shows the use of the device to image apiece of paper with fluorescent ink showing the word “Wound”. Inset f)shows imaging of fluorescent ink stained fingers, and detection of thecommon skin bacteria P. Acnes. The images from the cellular telephonemay be sent wirelessly to another cellular telephone, or wirelessly(e.g., via Bluetooth connectivity) to a personal computer for imagestorage and analysis. This demonstrates the capability of the device toperform real-time hand-held fluorescence imaging and wirelesstransmission to a remote site/person as part of a telemedicine/E-healthwound care infrastructure.

In order to demonstrate the capabilities of the imaging device in woundcare and other relevant applications, a number of feasibilityexperiments were conducted using the particular example described. Itshould be noted that during all fluorescence imaging experiments, theSony camera (Sony Cybershot DSC-T200 Digital Camera, Sony Corporation,North America) settings were set so that images were captured without aflash, and with the ‘Macro’ imaging mode set. Images were captured at 8megapixels. The flash was used to capture white light reflectanceimages. All images were stored on the xD memory card for subsequenttransfer to a personal computer for long-term storage and imageanalysis.

In one exemplary embodiment, white light reflectance and fluorescenceimages/movies captured with the device were imported into AdobePhotoshop for image analysis. However, image analysis software wasdesigned using MatLab™ (Mathworks) to allow a variety of image-basedspectral algorithms (e.g., red-to-green fluorescence ratios, etc.) to beused to extract pertinent image data (e.g., spatial and spectral data)for quantitative detection/diagnostic value. Image post-processing alsoincluded mathematical manipulation of the images.

In accordance with another exemplary embodiment, a handheld device forcollection of data from a wound includes a low-cost, consumer-grade,Super HAD™ charge-coupled device (CCD) sensor-based camera (ModelDSC-T900, Sony Corp., Japan), with a 35 to 140 mm equivalent 4× zoomlens housed in a plastic body and powered by rechargeable batteries(FIG. 5). A prototype of the handheld imaging device is shown in FIG. 5.Inset (a) is a front view of the prototype showing wound fluorescence(FL) image displayed in real time on the liquid-crystal display screenin high definition. Inset (b) is a back view of the prototype showingwhite light (WL) and 405-nm LED arrays providing illumination of thewound, while the FL emission filter is placed in front of the CCDsensor. The device is configured to collect high-resolution 12.1 Mpixelscolor WL and AF images (or videos) in real time (<1 s), which aredisplayed in red-green-blue (RGB) format on a 3.5-in. touch-sensitivecolor liquid-crystal display (LCD) screen of the device (FIG. 5). Thedevice includes broadband white light-emitting diodes (LEDs),electrically powered by a standard AC125V source, configured to provideillumination during WL imaging. The device further includes twomonochromatic violet-blue (λexc=405_20 nm) LED arrays (Model LZ4,LedEngin, San Jose, Calif.) to provide 4-W excitation light power duringFL imaging (bright, uniform illumination area ˜700 cm2 at 10 cm distancefrom skin surface). The WL and FL images are detected by ahigh-sensitivity CCD sensor mounted with a dual band FL filter(λemiss=500 to 550 and 590 to 690 nm) (Chroma Technologies Corp.,Vermont) in front of the camera lens to block excitation light reflectedfrom the skin surface. The device includes an emission filter configuredto spectrally separate tissue and bacteria AF. The device is configuredto display the spectrally separated tissue and bacterial AF as acomposite RGB image without image processing or color-correction, thusallowing the user to see the bacteria distribution within the anatomicalcontext of the wound and body site. The CCD image sensor is sensitiveacross ultraviolet (<400 nm), visible (400 to 700 nm), and near-infrared(700 to 900 nm) wavelengths to AF of tissues and bacteria, in theabsence of exogenous contrast agents.

In another exemplary embodiment, the handheld device is configured totake both white light images and fluorescent images incorporates amobile communication device, such as a smartphone, mobile phone, iPod,iPhone, or other such device having existing image-capturingcapabilities such as the CCD sensor. Although described herein withregard to usage with the iPod touch or iPhone, it should be understoodthat other platforms (e.g., Android, etc.) may be used. For example, asshown in FIG. 6A, the device incorporates an iPhone 4S. A mobile imagingdevice prototype is shown in FIG. 6A. Inset (a) shows a front view ofthe device, showing the optical components and battery holder of theaccessory adaptor, which is mounted onto a standard iPhone 4S smartphone. Inset (b) shows a back view of the device, showing the on/offswitch and the LCS viewing screen on which the WL and FL images areviewed by the user. White light imaging allows the user to capture animage of a patient wound and the fluorescence allows user to capture acorresponding image highlighting the presence of bacteria on the image.The display screen may range between about 4-inches (diagonal) and about7-inches (diagonal) widescreen display with Multi-Touch IPS technology.Other size displays may be used based on user needs. In one example, thedisplay quality settings are 1136×640-pixel resolution at 326 pixels perinch; 800:1 contrast ratio; and 500 cd/m2 max brightness. The displaymay have a fingerprint-resistant oleophobic coating. The resolution ofthe camera may be about 5 Megapixels and may have resolutions higherthan 5 Megapixels, such as up to about 24 Megapixels, depending uponavailability, amount of storage available, etc. The selection of thelens design allows the production of high quality images, specificallyin the red and green spectra. In one exemplary embodiment, afive-element lens is used (as iPod touch design). The user can tap tofocus video and/or still images. The camera has optimal performance inthe dark. The camera has an LED flash and shutter speeds are high.

As shown in FIGS. 6A and 6B, the exemplary embodiment of the handhelddevice integrates a consumer grade mobile phone camera with a customoptical platform. The image acquisition occurs on the mobile phonecamera and functions independently of the device housing, electronicsand optics. The images are displayed on the phone's LCD touch screen andare stored on the phone itself. The customized optical design includesone violet 405 nm LED positioned at a 45-degree angle to a dichroicmirror, which is fixed in front of the camera sensor. The dichroicmirror reflects violet light while transmitting all greater wavelengthsto produce fluorescence excitation illumination that is coaxial to thecamera sensor. A macro lens is situated in front of the camera sensor toallow for focused close up imaging of wounds (<10 cm). A specificcombination of excitation and emission filters are used to capture thered and green fluorescence data that is indicative of bacteria andconnective tissues respectively. The violet LED is powered by a standard9V battery, which is triggered by the user through an external powerswitch. A heat sink is attached to the back for the LED printed circuitboard with thermal paste to effectively transfer and dissipate the heatgenerated by the 5 W violet LED.

In accordance with this exemplary embodiment, the device housing may bemade by 3D printing. Other types of suitable structures are disclosedherein, and variations thereof will be understood by those of ordinaryskill in the art based on the present teachings. The housing provides ameans aligning the optical components with consumer grade camera andencasing both the electrical components used to drive the LED and thethermal solution while creating a user friendly and lightweighthand-held design. The adaptor is designed to slide onto the top of theiPhone 4s and fit snuggly around the phone to remain fixed in placeduring imaging. The adaptor is removable from the phone for white lightimaging. In accordance with another exemplary embodiment, the adaptormay be permanently affixed to the mobile communication device, such asthe iPhone 4s. In such an embodiment, a movable filter may be providedfor switching between white light imaging and fluorescent imaging, in amanner similar to that described with regard to embodiments of thehandheld device discussed in FIGS. 1 and 2.

To perform fluorescence imaging using the device, the user switches onthe violet LED using the toggle switch on the back of the device (FIG.6A). As the switch is moved to the ‘on’ position, the 9V battery sendspower to the LED drive, which modulates the current to drive the violetLED. The violet broad band LED, which is situated perpendicularly to theiPhone camera sensor, emits 405 nm light at the 45 degree dichroicmirror. The dichroic mirror reflects almost 100% of the light at the 405nm wavelength directly to the target. The tissues and bacteria absorbthe 405 nm photons from the violet LED and photons of a longerwavelength are then emitted by the bacteria and tissue to createfluorescence. A specific emission filter is placed in front of theiPhone camera sensor to control the wavelengths of photons that are ableto reach the camera sensor and effectively block the excitation light.The iPhone camera sensor captures an RGB image of the emitted photonswhere bacteria is displayed as red (e.g. S. aureus) or very brightbluish-green (e.g. Pseudomonas aruginosa) and healthy connective tissuesfrom skin or wound are captured by a green fluorescence signal. The userthen utilizes the fluorescence image (or video) stored on the mobilecommunication device, such as an iPhone, to determine where bacteria arelocated within and around a wound.

In one exemplary embodiment, a study using the handheld device describedherein tracked patient wounds over time. In the study, high resolutionWL PRODIGI images were taken of every wound at each visit. A disposablelength calibration scale (sticker) was placed near the wound during WLand FL imaging to track patient ID and date. A clinician marked thelocations of suspected clinically significant bacterial load on printedWL images. To preserve bacterial characteristics on the tissue, no swabwas taken until completion of subsequent FL imaging. This process took1-2 min per wound, and subsequent FL imaging took 1-2 min per wound. Thelocation(s) of positive red and/or green AF were marked on printedimages. The clinician swabbed each suspicious marked area using theLevine sampling method and swabs were sent for blinded microbiologytesting. Patients were treated and discharged according to standardprotocols. FL spectroscopy was used in some cases to characterize AFareas in/around the wound. Spectra were compared on a location basiswith microbiology results. A complete data file for each patient's visit(CSS, WL and FL images, spectroscopy and microbiology) were stored in anelectronic database according to Good Clinical Practice guidelines.

In a second part of the study, three sequential 2-month arms were used:non-guided treatment (control), FL-guided treatment and non-guidedtreatment (control). In the first 2-month phase, wounds were assessedweekly by CSS and then treated at the discretion of the clinical teamusing best practice methods (ultrasonic and/or scalpel wounddebridement, topical/systemic antibiotics, saline wash, dry oranti-microbial dressings or iodine). Corresponding WL and FL images weretaken of each wound pre- and post-treatment as described previously. 2month evaluation periods were selected based on established clinicaldata for venous leg ulcers showing that this is sufficient to detect areliable and meaningful change in wound area, as a predictive indicatorof healing. Wound swabs were collected by FL guidance. Clinicians wereblinded to FL images during this first (control) phase. During thesubsequent 2 month phase, wound assessment was performed normally butclinicians were shown FL images of the wound during treatment.

During the final 2 month phase, WL and FL imaging were performed andswabs were collected, with clinician blinding to the FL results duringtreatment delivery. Importantly, while the clinicians understood andcould remember the meaning and characteristics of the red and greenfluorescence signals, respectively, blinding them during treatmentdelivery in the control periods was possible because the fluorescenceresults for each wound examination and each patient were different.Thus, in the absence of real-time fluorescence guidance during woundtreatment, previous knowledge of fluorescence characteristics did notsubstantively influence the treatment decisions during the controlperiods. WL and FL images were also taken after each treatment toanalyze wound area.

Four blinded, trained clinical and/or research staff membersindependently measured the average wound size on WL images using digitaltracing (MATLAB v.7.9.0, The MathWorks, Mass., USA). The observersmeasured the wounds in separate sessions with a minimum of 7 daysbetween sessions to minimize memory effect. An adhesive scale bar placedadjacent to the wound during imaging provided accurate lengthcalibration within +0.5 mm. Room lights remained on during WL imaging,but were turned off during FL imaging. WL and FL images were collectedwith the handheld device held/positioned 10-15 cm from the wound. Allimaging parameters (distance, exposure time, ISO setting, white balance,etc.) were kept constant between visits. For distances less than 5 cmfrom a wound (small diameter wounds), the camera's built-in macro modewas used. Automatic focusing allowed rapid (˜1s) image acquisition.Images (or video) were captured in real-time and stored on the camera'smemory card. Switching between WL and FL modes was substantiallyinstantaneous using a built in “toggle switch.” Devices weredecontaminated between uses with 70% ethyl alcohol.

WL and AF images were transferred to a laptop. Regions of interest(ROIs) were identified from individual 1024×1024 pixel FL images of eachwound at each clinic visit. RGB images were separated into individualchannels. The green and red channels of the RGB image wererepresentative of the true tissue and bacterial AF signals detected invivo. To quantify bacterial levels from individual FL images, thefollowing image processing procedures were used. Briefly, individualgreen and red image channels from each RGB image were converted togreyscale (the blue channel was not used) and pixels whose greyscaleintensity was above a given histogram threshold (selected to reduce thebackground noise of the raw image) were counted. A red color mask forred FL bacteria was created by finding the local maxima in the colorrange 100-255 greyscale. Then, an inverted green color mask was used toremove the green FL. All pixels with red FL (above the histogramthreshold) were binarized and the sum of all “1” pixels was calculated.This was repeated for the green channel of each image. These data gavean estimate of the amount of red (or green) bacteria in each image. Thenumber of FL pixels was converted into a more useful pixel area measure(cm2) using the adhesive length calibration stickers, thereby providingthe total amount of fluorescent bacteria as an area measurement.

Tissue AF produced by endogenous collagen or elastin in the skinappeared as green FL, and clinically-relevant bacterial colonies (e.g.Staphylococcus aureus) appeared as red FL (caused by endogenousporphyrins. Some bacteria (e.g. Pseudomonus aeruginosa) produced ablue-green signal, due to siderophores/pyoverdins, which wasdifferentiated spectrally and texturally from dermis AF using imageanalysis software. WL and FL images were collected in less than 1 secondby the high-sensitivity CCD sensor mounted with a dual band FL filter(λ_(emiss)=500-550 and 590-690 nm) (Chroma Technologies Corp, VT, USA).The CCD image sensor was sensitive across a broad wavelength range of˜300-800 nm. PRODIGI integrated easily into the routine clinical workflow. By combining tissue FL with bacterial FL in a single compositeimage, the clinician instantly visualized the distribution and extent ofthe bacterial load within the anatomical context of the wound and bodysite. Typically, FL imaging added approximately 1-3 minutes/patient tothe wound assessment routine, depending on the number of wounds and theduration of FL-guided swabbing.

AF imaging detected clinically significant bacterial load in 85% ofwound peripheries missed by conventional methods. Thus, the Levinemethod for swabbing only the wound bed may be insufficient, possiblyresulting in antibacterial treatment being inappropriately withheld.However, modifying standard sampling practices to include swabbing ofthe wound periphery of all wounds would be impractical and costly. AFimaging could help clinicians decide if and where wound margins requiresampling. The handheld imaging device also identified clinicallysignificant bioburden in surrounding locations close to wounds, whichrepresent sites of potential re-infection, where traditional methods donot examine or swab.

Identifying and quantitating wound bacterial burden is an importantdeterminant of infection and healing. Data on the visualization andquantitative tracking of bacterial load led to the identification of anew, simple method for image-guided debridement and topical applicationof antibiotic and antiseptic, which minimizes unnecessary trauma to thewound boundary and maximizes the contribution of debridement to reducingbacterial burden. Every wound has the potential for infection, butdistinguishing true infection from critical colonization by bestpractice methods remains challenging and arbitrary, and can lead toover- and under-treatment.

Multiple variables including host response, local and systemic factors,malperfusion, immunosuppression, diabetes, and medications affect therisk of infection. Critically colonized wounds can be difficult todiagnose because they do not always display classical signs of infectionor clearly elevated levels of bioburden. Indeed, the clinical relevanceof differentiating critically colonized wounds from infected woundsremains controversial. Identifying a high bacterial load in asymptomaticpatients before infection occurs using AF imaging may help preventinfections by prompting aggressive treatment. If a bacterial infectionis suspected, antibiotic selection could be guided by the establishedclinical principles and by AF identification of heavy bacterial burdenand differentiation between Gram negative P. aeruginosa and Grampositive S. aureus.

In another exemplary embodiment, image analysis may be carried out onthe handheld device or WL and FL images may be transferred to a laptopfor image processing. Image analysis and processing of image data may beperformed using a processor of the handheld device, and the results ofsuch analyses may be displayed on the display of the handheld device.

The following two programs may be used for image processing (forexample, analysis of the data collected by the exemplary device usingthe Super HAD™ charge-coupled device (CCD) sensor-based camera (ModelDSC-T900)) and portions of these processes are illustrated in FIGS. 7Aand 7B: MATLAB software (Version 7.9.0, The MathWorks, Mass.) using acustom-written program and ImageJ Software (Version 1.45n). In theMATLAB program, regions of interests (ROIs) are identified fromindividual 1024×1024 pixel FL images of each wound. RGB images areseparated into individual channels. Green (500 to 550 nm emission) andred AF (>590 nm) from tissue components and bacteria, respectively,detected by the CCD sensor are naturally aligned spectrally with the redand green filters on the Sony CCD image sensor. Thus, the green and redchannels of the RGB image displayed on the handheld device's LCD screenare representative of the true tissue and bacterial AF signals detectedin vivo. To quantify bacterial levels from individual FL images, thefollowing image processing procedures may be used. Briefly, individualgreen and red image channels from each RGB image are converted to grayscale (the blue channel is not used) and pixels whose gray scaleintensity is above a given histogram threshold (selected to reduce thebackground noise of the raw image) are counted. In certain embodiments,it is possible the blue channel would be used, for example, when imagingthe amount of 405 nm excitation light that is absorbed by tissues/bloodwhen imaging tissue vascularity/perfusion.

A red color mask for red FL bacteria is created by finding the localmaxima in the color range 100 to 255 gray scale. Then, an inverted greencolor mask is used to remove the green FL. All pixels with red FL (abovethe histogram threshold) are binarized and the sum of all “1” pixels iscalculated. This is repeated for the green channel of each image. Thesedata give an estimate of the amount of red bacteria in each image. Thenumber of FL pixels is converted into a more useful pixel area measure(cm2) by applying a ruler on the pixel image, thereby providing thetotal amount of fluorescent bacteria as an area measurement (cm2). Thesizes of the wounds may be traced and measured similarly by convertingpixel areas to cm2 of the circled wound area on the WL images. Theresolution of the FL images is sufficient to localize bacteria based onregions of FL. ImageJ software may be used to separate FL images intored, green, and blue channels using the built-in batch processingfunction “Split Channels” located within the image menu and colorsubmenu of the camera. Each resulting channel is displayed and saved ingray scale. For further analysis, an ROI may be identified in eachcorresponding red, green, and blue channel image. Under the built-inanalysis menu, the “Set Measurement” function may be used to select andmeasure the following measurement parameters for each color channelimage: pixel area, min. and max. gray scale intensity values, and meangray intensity values. The average red channel intensity value may bedetermined as (bacterial) FL intensity per square pixel in each redchannel image and then used for data analysis and comparison.

In one exemplary embodiment, a mouse skin wound model was used tocorrelate wound status with the progression of bacterial infection (n=5;8 to 12 weeks; NCRNU-F). Correlation was based on data obtained usingthe exemplary handheld device described above, which incorporates theSuper HAD™ charge-coupled device (CCD) sensor-based camera (ModelDSC-T900. Daily WL and FL images were taken of the wounds as they becameinfected over time. Antibacterial treatment (topical Mupirocin threetimes daily, for a total of 1 day) was applied to the wound site whenthe red FL intensity peaked. The anti-microbial effect of the treatmentwas monitored over time using the handheld device to acquire daily WLand FL images of the wound after treatment. The wounds were monitoredfor a total of 10 days (see FIG. 8), after which the mice weresacrificed. Bacterial amounts from FL images and wound size from WLimages were quantified using the MATLAB program described above, andcompared over time to determine the wound healing status.

FIG. 8 shows representative WL and FL images for a single mouse trackedover 10 days. Inset (a) provides images taken with the prototype deviceand showing the two equal-sized wounds on both sides of the spine. Theright wound was inoculated with S. aureus in PBS and the left wound wasinoculated with PBS only (control). The top row shows WL images, themiddle row shows FL images, and the bottom row shows MATLAB quantifiedimages, corresponding to bacterial areas and intensities. The FL imagingdata demonstrated a significant increase in bacterial FL intensity inthe wound inoculated with S. aureus, compared with the control wound,peaking on day 6. Mupirocin (day 7, red arrow) significantly decreasedbacterial FL on day 8 to almost zero, indicating treatment effect.Bacteria increased again on days 9 and 10. Inset (b) provides a graphshowing quantitative changes in bacterial load from FL images obtainedin inset (a).

In accordance with another exemplary embodiment, BLI can be used tomeasure the absolute amount of bacteria in vivo, because it is one ofthe most sensitive and reliable screening tools for determiningbacterial load. BLI collects the light emitted from the enzymaticreaction of luciferase and luciferin and therefore does not requireexcitation light. FL imaging using the handheld device (without anyexogenous FL contrast agent administration) and BLI imaging ofinoculated S. aureus bacteria were tracked over time and the FL and BLIintensities were compared (see FIG. 9) (n=7). The bacterial BLI signaldid not contribute to the FL signal detected by the handheld device'sconsumer grade-CCD camera. Gram-positive bioluminescent S. aureus-Xen8.1from the parental strain S. aureus 8325-4 (Caliper) was grown tomid-exponential phase the day before pathogen inoculation. Bacteria withthe BLI cassette produce the luciferase enzyme and its substrate(luciferin), thereby emitting a 440 to 490 nm bioluminescent signal whenmetabolically active (FIG. 9). The bacteria (1010CFU) were suspended in0.5 mL of PBS and injected into the wounds of female athymic nude mice(n=7; 8 to 12 weeks; NCRNU-F Homozygous). To detect S. aureusbioluminescence, BLI images of the wound were acquired before,immediately after, and 1, 2, 3, 4, 5, 6, and 7 days postinoculationinside the dark chamber of the Xenogen IVIS Spectrum Imaging System 100(Caliper, Mass.), using an exposure time of 10 s. BLI images werecaptured using Living Image In Vivo Imaging software (Caliper, Mass.).ROIs were digitally circumscribed over the wound and the totalluminescence intensity counts were measured within the ROIs for eachtime point imaged. The absolute amount of bacteria measured from the BLIsignals was tested for correlation with the corresponding FL signals onthe FL images taken over time of the same wound using the handhelddevice (as described above).

FIG. 9 provides preclinical data which show that pathogenic bacterialautofluorescence (AF) intensity correlates with bacterial load in vivo.Inset (a) shows a time course prototype device mobile images of skinwounds in a mouse prior to and after inoculation with bioluminescent S.aureus-Xen8.1 (10¹⁰ CFU in 30 μL PBS). Representative WL (top row), AF(middle row), and bioluminescence (bottom row) images are shown for eachtime point to 7 days after inoculation in a wounded mouse. BLI imaginggives absolute bacterial amount in vivo. Red arrows show when thetegaderm bandage was exchanged, causing some bacteria to be removed fromthe surface. Inset (b) shows average red FL from S. aureus-Xen8.1 (n=7nude mice) shown as a function of time demonstrating an increase indaily S. aureus bacterial FL (calculated from red channel of RGB imagesusing ImageJ software). At days 2 and 7, tegaderm bandages wereexchanged as per animal protocol. Average bacterial FL peaked at day 4postinoculation. Inset (c) illustrates a corresponding time coursebioluminescence data (calculated from ROI) show similar increase andpeaking at day 4 in total bacterial load in the wound. Data indicatestrong positive correlation (Pearson correlation coefficient r=0.6889)between total bacterial AF in a wound and the bacterial load in vivo.Standard errors are shown. Scale bars: (a) WL 1.5 cm and AF, BLI 1 cm.

Imaging of Bacteriological Samples

The imaging device may be useful for imaging and/or monitoring inclinical microbiology laboratories. The device may be used forquantitative imaging of bacterial colonies and quantifying colony growthin common microbiology assays. Fluorescence imaging of bacterialcolonies may be used to determine growth kinetics. Software may be usedto provide automatic counting of bacterial colonies.

To demonstration the utility of the device in a bacteriology/culturelaboratory, live bacterial cultures were grown on sheep's blood agarplates. Bacterial species included streptococcus pyogenes, serratiamarcescens, staphylococcus aureus, staphylococcus epidermidis,escherichia coli, and pseudomonas aeruginosa (American Type CultureCollection, ATCC). These were grown and maintained under standardincubation conditions at 37° C. and used for experimentation when during‘exponential growth phase’. Once colonies were detected in the plates(˜24 h after inoculation), the device was used to image agar platescontaining individual bacterial species in a darkened room. Usingviolet/blue (about 405 nm) excitation light, the device was used toimage both combined green and red autofluorescence (about 490-550 nm andabout 610-640 nm emission) and only red autofluorescence (about 635+/−10nm, the peak emission wavelength for fluorescent endogenous porphyrins)of each agar plate. Fluorescence images were taken of each bacterialspecies over time for comparison and to monitor colony growth.

Reference is now made to FIG. 10. Inset a) shows the device being usedto image live bacterial cultures growing on sheep's blood agar plates todetect bacterial autofluorescence. Inset b) shows the image ofautofluorescence emitted by pseudomonas aruginosa. The device may alsobe used to detect, quantify and/or monitor bacterial colony growth overtime using fluorescence, as demonstrated in inset c) with fluorescenceimaging of the growth of autofluorescent staphylococcus aureus on anagar plate 24 hours after innoculation. Note the presence of distinctsingle bacterial colonies in the lower image. Using violet/blue (e.g.,405 nm) excitation light, the device was used to detect both combinedgreen and red (e.g., 490-550 nm+610-640 nm) and only red (e.g., 635+/−10nm, the peak emission wavelength for fluorescent endogenous porphyrins)emission autofluorescence from several live bacterial species includingstreptococcus pyogenes, shown in inset d); serratia marcescens, shown ininset e); staphylococcus aureus, shown in inset f); staphylococcusepidermidis, shown in inset g); escherichia coli, shown in inset h); andPseudomonas aeruginosa, shown in inset i). Note that theautofluorescence images obtained by the device of the bacterial coloniesmay provide useful image contrast for simple longitudinal quantitativemeasurements of bacterial colonization and growth kinetics, as well as ameans of potentially monitoring response to therapeutic intervention,with antibiotics, photodynamic therapy (PDT), low level light therapy,hyperbaric oxygen therapy (HOT), or advanced wound care products, asexamples.

High spatial resolution of the camera detector combined with significantbacterial autofluorescence signal-to-noise imaging with the deviceallowed detection of very small (e.g., <1 mm diameter) colonies. Thedevice provided a portable and sensitive means of imaging individualbacterial colonies growing in standard agar plates. This provided ameans to quantify and monitor bacterial colony growth kinetics, as seenin inset c), as well as potentially monitoring response to therapeuticintervention, with antibiotics or photodynamic therapy (PDT) asexamples, over time using fluorescence. Therefore, the device may serveas a useful tool in the microbiology laboratory.

FIG. 11 shows an example of the use of the imaging device in inset a)standard bacteriology laboratory practice. Inset b) Here, fluorescenceimaging of a Petri dish containing Staphylococcus aureus combined withcustom proprietary image analysis software allows bacterial colonies tobe counted rapidly, and here the fluorescence image of the culture dishshows ˜182 (+/−3) colonies (bright bluish-green spots) growing on agarat 37° C. (about 405 nm excitation, about 500-550 nm emission (green),about >600 nm emission (red)).

In addition to providing detecting of bacterial species, the device maybe used for differentiating the presence and/or location of differentbacterial species (e.g., Staphylococcus aureus or Pseudomonasaeguginosa), for example in wounds and surrounding tissues. This may bebased on the different autofluorescence emission signatures of differentbacterial species, including those within the 490-550 nm and 610-640 nmemission wavelength bands when excited by violet/blue light, such aslight around 405 nm. Other combinations of wavelengths may be used todistinguish between other species on the images. This information may beused to select appropriate treatment, such as choice of antibiotic.

Such imaging of bacteriology samples may be applicable to monitoring ofwound care.

Use in Monitoring of Wound Healing

The device may be scanned above any wound (e.g., on the body surface)such that the excitation light may illuminate the wound area. The woundmay then be inspected using the device such that the operator may viewthe wound in real-time, for example, via a viewer on the imaging deviceor via an external display device (e.g., heads-up display, a televisiondisplay, a computer monitor, LCD projector or a head-mounted display).It may also be possible to transmit the images obtained from the devicein real-time (e.g., via wireless communication) to a remote viewingsite, for example for telemedicine purposes, or send the images directlyto a printer or a computer memory storage. Imaging may be performedwithin the routine clinical assessment of patient with a wound.

Prior to imaging, fiduciary markers (e.g., using an indeliblefluorescent ink pen) may be placed on the surface of the skin near thewound edges or perimeter. For example, four spots, each of a differentfluorescent ink color from separate indelible fluorescent ink pens,which may be provided as a kit to the clinical operator, may be placednear the wound margin or boundary on the normal skin surface. Thesecolors may be imaged by the device using the excitation light and amultispectral band filter that matches the emission wavelength of thefour ink spots. Image analysis may then be performed, by co-registeringthe fiduciary markers for inter-image alignment. Thus, the user may nothave to align the imaging device between different imaging sessions.This technique may facilitate longitudinal (i.e., over time) imaging ofwounds, and the clinical operator may therefore be able to image a woundover time without need for aligning the imaging device during everyimage acquisition.

In addition, to aid in intensity calibration of the fluorescence images,a disposable simple fluorescent standard ‘strip’ may be placed into thefield of view during wound imaging (e.g., by using a mild adhesive thatsticks the strip to the skin temporarily). The strip may be impregnatedwith one or several different fluorescent dyes of varying concentrationswhich may produce pre-determined and calibrated fluorescence intensitieswhen illuminated by the excitation light source, which may have single(e.g., 405 nm) or multiple fluorescence emission wavelengths orwavelength bands for image intensity calibration. The disposable stripmay also have the four spots as described above (e.g., each of differentdiameters or sizes and each of a different fluorescent ink color with aunique black dot placed next to it) from separate indelible fluorescentink pens. With the strip placed near the wound margin or boundary on thenormal skin surface, the device may be used to take white light andfluorescence images. The strip may offer a convenient way to takemultiple images over time of a given wound and then align the imagesusing image analysis. Also, the fluorescent ‘intensity calibration’strip may also contain an added linear measuring apparatus, such as aruler of fixed length to aid in spatial distance measurements of thewounds. Such a strip may be an example of a calibration target which maybe used with the device to aid in calibration or measuring of imageparameters (e.g., wound size, fluorescence intensity, etc.), and othersimilar calibration target may be used.

It may be desirable to increase the consistency of imaging results andto reproduce the distance between the device and the wound surface,since tissue fluorescence intensity may vary slightly if the distancechanges during multiple imaging sessions. Therefore, in an embodiment,the device may have two light sources, such as low power laser beams,which may be used to triangulate individual beams onto the surface ofthe skin in order to determine a fixed or variable distance between thedevice and the wound surface. This may be done using a simply geometricarrangement between the laser light sources, and may permit the clinicaloperator to easily visualize the laser targeting spots on the skinsurface and adjust the distance of the device from the wound duringmultiple imaging sessions. Other methods of maintaining a constantdistance may include the use of ultrasound, or the use of a physicalmeasure, such as a ruler, or a range finder mechanism.

Use in White Light Imaging

The device may be used to take white light images of the total woundwith normal surrounding normal tissues using a measuring apparatus(e.g., a ruler) placed within the imaging field of view. This may allowvisual assessment of the wound and calculation/determination ofquantitative parameters such as the wound area, circumference, diameter,and topographic profile. Wound healing may be assessed by planimetricmeasurements of the wound area at multiple time points (e.g., atclinical visits) until wound healing. The time course of wound healingmay be compared to the expected healing time calculated by the multipletime point measurements of wound radius reduction using the equationR=√A/π (R, radius; A, planimetric wound area; π, constant 3.14). Thisquantitative information about the wound may be used to track andmonitor changes in the wound appearance over time, in order to evaluateand determine the degree of wound healing caused by natural means or byany therapeutic intervention. This data may be stored electronically inthe health record of the patient for future reference. White lightimaging may be performed during the initial clinical assessment of thepatient by the operator.

Use in Autofluorescence Imaging

The device may be designed to detect all or a majority of tissueautofluorescence (AF). For example, using a multi-spectral band filter,the device may image tissue autofluorescence emanating from thefollowing tissue biomolecules, as well as blood-associated opticalabsorption, for example under 405 nm excitation: collagen (Types I, II,III, IV, V and others) which appear green, elastin which appearsgreenish-yellow-orange, reduced nicotinamide adenine dinucleotide(NADH), flavin adenine dinucleotide (FAD), which emit a blue-greenautofluorescence signal, and bacteria/microorganisms, most of whichappear to have a broad (e.g., green and red) autofluorescence emission.

Image analysis may include calculating a ratio of red-to-green AF in theimage. Intensity calculations may be obtained from regions of interestwithin the wound images. Pseudo-coloured images may be mapped onto thewhite light images of the wound.

Examples in Wound Healing

Reference is now made to FIG. 12. The device was tested in a model ofwounds contaminated with bacteria. For this, pig meat, with skin, waspurchased from a butcher. To simulate wounds, a scalpel was used to makeincisions, ranging in size from 1.5 cm² to 4 cm² in the skin, and deepenough to see the muscle layer. The device was used to image some meatsamples without (exogenous) addition of bacteria to the simulatedwounds. For this, the meat sample was left at room temperature for 24 hin order for bacteria on the meat to grow, and then imaging wasperformed with the device using both white light reflectance andautofluorescence, for comparison.

To test the ability of the device to detect connective tissues andseveral common bacteria present in typical wounds, a sample of pig meatwith simulated wounds was prepared by applying six bacterial species toeach of six small 1.5 cm² wound incision sites on the skin surface:streptococcus pyogenes, serratia marcescens, staphylococcus aureus,staphylococcus epidermidis, escherichia coli, and pseudomonasaeruginosa. An additional small incision was made in the meat skin,where no bacteria were added, to serve as a control. However, it wasexpected that bacteria from the other six incisions sites would perhapscontaminate this site in time. The device was used to image thebacteria-laden meat sample using white light reflectance and violet/bluelight-induced tissue autofluorescence emission, using both a dualemission band (450-505 nm and 590-650 nm) emission filter and a singleband (635+/−10 nm) emission filter, on the left and a single band filterover the course of three days, at 24 h time intervals, during which themeat sample was maintained at 37° C. Imaging was also performed on thestyrofoam container on which the meat sample was stored during the threedays.

FIG. 12 shows the results of the device being used for non-invasiveautofluorescence detection of bacteria in a simulated animal woundmodel. Under standard white light imaging, bacteria were occult withinthe wound site, as shown in inset a) and magnified in inset b). However,under violet/blue excitation light, the device was capable of allowingidentification of the presence of bacteria within the wound site basedon the dramatic increase in red fluorescence from bacterial porphyrinsagainst a bright green fluorescence background from connective tissue(e.g., collagen and elastins) as seen in inset c) and magnified in insetd). Comparison of inset b) and inset d) shows a dramatic increase in redfluorescence from bacterial porphyrins against a bright greenfluorescence background from connective tissue (e.g., collagen andelastins). It was noted that with autofluorescence, bacterial colonieswere also detected on the skin surface based on their green fluorescenceemission causing individual colonies to appear as punctuate green spotson the skin. These were not seen under white light examination.Fluorescence imaging of connective tissues aided in determining thewound margins as seen in inset e) and inset f), and some areas of theskin (marked ‘*’ in c) appeared more red fluorescent than other areas,potentially indicating subcutaneous infection of porphyrin-producingbacteria. Insets e) and f) also show the device detecting redfluorescent bacteria within the surgical wound, which are occult underwhite light imaging.

The device mapped biodistribution of bacteria within the wound site andon the surrounding skin and thus may aid in targeting specific tissueareas requiring swabbing or biopsy for microbiological testing.Furthermore, using the imaging device may permit the monitoring of theresponse of the bacterially-infected tissues to a variety of medicaltreatments, including the use of antibiotics and other therapies, suchas antibiotics, wound debridement, wound cleaning, photodynamic therapy(PDT), hyperbaric oxygen therapy (HOT), low level light therapy, oranti-matrix metalloproteinase (MMP). The device may be useful forvisualization of bacterial biodistribution at the surface as well aswithin the tissue depth of the wound, and also for surrounding normaltissues. The device may thus be useful for indicating the spatialdistribution of an infection.

Examples

Reference is now made to FIG. 13. As an example, the imaging device maybe used clinically to determine the healing status of a chronic woundand the success of wound debridement. For example, a typical foot ulcerin a person with diabetes is shown in the figure, with (i) thenonhealing edge (i.e., callus) containing ulcerogenic cells withmolecular markers indicative of healing impairment and (ii)phenotypically normal but physiologically impaired cells, which can bestimulated to heal. Despite a wound's appearance after debridement, itmay not be healing and may need to be evaluated for the presence ofspecific molecular markers of inhibition and/or hyperkeratotic tissue(e.g., c-myc and β-catenin). Using the imaging device in combinationwith exogenous fluorescently labeled molecular probes against suchmolecular targets, the clinician may be able to determine the in situexpression of molecular biomarkers. With the device, once a wound isdebrided, fluorescence imaging of the wound area and image analyses mayallow biopsy targeting for subsequent immunohistochemistry and this maydetermine whether the extent of debridement was sufficient. If theextent of debridement was insufficient, as shown in the lower leftdiagram, cells positive for c-myc (which appears green) and nuclearβ-catenin (which appears purple) may be found based on theirfluorescence, indicating the presence of ulcerogenic cells, which mayprevent the wound from healing properly and indicate that additionaldebridement is necessary. Lack of healing may also be demarcated by athicker epidermis, thicker cornified layer, and presence of nuclei inthe cornified layer. If the debridement was successful, as in the lowerright lower diagram, no staining for c-myc or β-catenin may be found,indicating an absence of ulcerogenic cells and successful debridement.These markers of inhibition may be useful, but the goal is actualhealing as defined by the appearance of new epithelium, decreased areaof the wound, and no drainage. This information may be collected usingthe fluorescence imaging device and stored electronically in thepatient's medical record, which may provide an objective analysiscoupled with pathology and microbiology reports. By comparing expectedhealing time with actual healing (i.e., healing progress) time using theimaging device, adaptive treatment strategies may be implemented on aper-patient basis.

FIG. 14 shows an example of the use of the device for imaging woundhealing of a pressure ulcer. Inset a) White light image taken with thedevice of the right foot of a diabetic patient with a pressure ulcer isshown. Inset b) Corresponding fluorescence image shows the bright redfluorescence of bacteria (bacteriology results confirmed presence ofheavy growth of Staphylococcus aureus) which are invisible understandard white light examination (yellow arrows). Note the heavy growthof Staphylococcus aureus bacteria around the periphery of thenon-healing wound (long yellow arrow). Insets c-d) Show thespectrally-separated (unmixed) red-green-blue images of the rawfluorescence image in inset b), which are used to producespectrally-encoded image maps of the green (e.g. collagen) and red (e.g.bacteria) fluorescence intensities calculated using mathematicalalgorithms and displayed in false color with color scale. Insets f-g)show examples of image-processing methods used enhance the contrast ofthe endogenous bacterial autofluorescence signal by calculating thered/green fluorescence intensity ratio to reveal the presence andbiodistribution of bacteria (red-orange-yellow) within and around theopen wound. These data illustrate the ability to use custom orcommercially-available image-analysis software to mathematically analyzethe fluorescence images obtained by the device and display them in ameaningful way for clinical use, and this may be done in real-time.(Scale bar 1 cm).

FIG. 15 shows an example of the use of the device for imaging a chronicnon-healing wound. Inset a) White light image taken with the device ofthe left breast of a female patient with Pyoderma gangrenosum, shows achronic non-healing wound (blue arrow) and a healed wound (red arrow).Bacteria typically cannot be visualized by standard white lightvisualization used in conventional clinical examination of the wounds.Inset b) Corresponding fluorescence image of the same wounds (in thisexample, using 405 nm excitation, 500-550 nm emission (green), >600 nmemission (red)) is shown. Note that the non-healed wound appears darkcolored under fluorescence (mainly due to blood absorption of theexcitation and fluorescence emission light), while bacteria appear aspunctuate bright red spots in the healed wound (red arrow). Underfluorescence, normal surrounding skin appears cyan-green due toendogenous collagen fluorescence (405 nm excitation). By contrast, thenon-healed wound (blue arrow) appears to have a band of very bright redfluorescence around the wound border, confirmed with swab cultures(bacteriology) to contain a heavy growth of Staphylococcus aureus (withfew Gram positive bacilli and rare Gram positive cocci, confirmed bymicroscopy). Inset c) White light image of the healed wound in insets a,b) and d) corresponding fluorescence image showing bright redfluorescence from bacteria (pink arrows), which are occult under whitelight. Inset e) White light and inset f) corresponding fluorescenceimages of the non-healed breast wound. Note that bacteria(Staphylococcus aureus) appear to be mainly localized around theedge/boundary of the wound (yellow arrow), while less bacteria arelocated within the wound (X), determined by the biodistribution ofbacteria directly visualized using fluorescence imaging, but invisibleunder white light (black arrow, e). (Scale bar in cm).

FIG. 16 further illustrates imaging of a chronic non-healing wound usingan example of the imaging device. Inset a) White light image taken withthe device of left breast of female patient with Pyoderma gangrenosum,showing chronic non-healing wound (blue arrow) and healed wound (bluearrow). Bacteria cannot be visualized by standard white lightvisualization used in clinical examination of the wounds. Inset b)Corresponding fluorescence image of the same wounds (405 nm excitation,500-550 nm emission (green), >600 nm emission (red)). While the nippleappears to be normal under white without obvious contamination ofbacteria, fluorescence imaging shows the presence of bacteria emanatingfrom the nipple ducts. Swabs of the nipple showed bacteria wereStaphylococcus epidermidis (Occasional growth found on culture). (Scalebar in cm)

FIG. 17 shows a central area and border of a chronic non-healing woundimaged using the imaging device. a) White light image taken with thedevice of left breast of female patient with Pyoderma gangrenosum,showing the central area and border of a chronic non-healing wound.Inset a) White light and inset b) corresponding fluorescence images ofthe non-healed breast wound (405 nm excitation, 500-550 nm emission(green), >600 nm emission (red)). Note that bacteria (Staphylococcusaureus; shown by bacterial swabbing) appear to be mainly localizedaround the edge/boundary of the wound, while less bacteria are locatedwithin the wound (X), determined by the biodistribution of bacteriadirectly visualized using fluorescence imaging, but invisible underwhite light. (Scale bar in cm).

FIG. 18 shows further images of a chronic non-healing wound using theimaging device. Inset a) White light image taken with the device of leftbreast of female patient with Pyoderma gangrenosum, showing chronicnon-healing wound. Bacteria cannot be visualized by standard white lightvisualization used in clinical examination of the wounds. Inset b)Corresponding fluorescence image of the same wound (405 nm excitation,500-550 nm emission (green), >600 nm emission (red)). Fluorescenceimaging shows the presence of bacteria around the wound edge/borderpre-cleaning inset (b) and post-cleaning inset (c). In this example,cleaning involved the use of standard gauze and phosphate bufferedsaline to wipe the surface the wound (within and without) for 5 minutes.After cleaning, the red fluorescence of the bacteria is appreciablydecreased indicating that some of the red fluorescent bacteria mayreside below the tissue surface around the edge of the wound. Smallamounts of bacteria (red fluorescent) remained within the wound centerafter cleaning. This illustrates the use of the imaging device tomonitor the effects of wound cleaning in real-time. As an additionalexample, inset d) shows a white light image of a chronic non-healingwound in the same patient located on the left calf. Inset e) Shows thecorresponding fluorescence images pre-cleaning inset (e) andpost-cleaning inset (f). Swabbing of the central area of the woundrevealed the occasional growth of Staphylococcus aureus, with a heavygrowth of Staphylococcus aureus at the edge (yellow arrow). Cleaningresulted in a reduction of the fluorescent bacteria (Staphylococcusaureus) on the wound surface as determined using the handheld opticalimaging device. The use of the imaging device resulted in the real-timedetection of white light-occult bacteria and this allowed an alterationin the way the patient was treated such that, following fluorescenceimaging, wounds and surrounding (bacteria contaminated) were eitherre-cleaned thoroughly or cleaned for the first time because of de novodetection of bacteria. Also, note the use of a disposable adhesivemeasurement-calibration ‘strip’ for aiding in imaging-focusing and this“strip” may be adhered to any part of the body surface (e.g., near awound) to allow wound spatial measurements. The calibration strip mayalso be distinctly fluorescent and may be used to add patient-specificinformation to the images, including the use of multiple exogenousfluorescent dyes for “barcoding” purposes—the information of which canbe integrated directly into the fluorescence images of wounds. (Scalebar in cm).

FIG. 19 illustrates use of the imaging device for monitoring woundhealing over time. The imaging device is used for tracking changes inthe healing status and bacterial biodistribution (e.g. contamination) ofa non-healing chronic wound from the left breast of female patient withPyoderma gangrenosum. White light images (see column showing insets a-m)and corresponding fluorescence images of the healed wound (see columnshowing insets b-n) and of the chronic non-healing wound (see columnshowing insets c-o) are shown over the course of six weeks. (405 nmexcitation, 500-550 nm emission (green), >600 nm emission (red)), takenusing the imaging device under both white light and fluorescence modes.In the column of insets b-n), the presence of small bright redfluorescence bacterial colonies are detected (yellow arrows), and theirlocalization changes over time within the healed wound. Bacterial swabsconfirmed that no bacteria were detected on microscopy and no bacterialgrowth was observed in culture. In the column of insets c-o), bycontrast, the non-healed wound has a band of very bright redfluorescence around the wound border, confirmed with swab cultures(bacteriology) to contain a heavy growth of Staphylococcus aureus (withfew Gram positive bacilli and rare Gram positive cocci, confirmed bymicroscopy), which changes in biodistribution over time (i.e., seecolumn of insets c-o). These data demonstrate that the imaging devicemay yield real-time biological and molecular information as well as beused to monitor morphological and molecular changes in wounds over time.

FIG. 20 shows another example of the use of the device for monitoringwound status over time. The imaging device is used tracking changes inthe healing status and bacterial biodistribution (e.g. contamination) ofa wound from the left calf of 21 year old female patient with Pyodermagangrenosum. White light images (see column of insets a-i) andcorresponding fluorescence images (see column of insets b-j) of a woundbeing treated using hyperbaric oxygen therapy (HOT) are shown over thecourse of six weeks. (Fluorescence parameters: 405 nm excitation,500-550 nm emission (green), >600 nm emission (red)). Column of insetsa-i) White light images reveal distinct macroscopic changes in the woundas it heals, indicated by the reduction in size over time (e.g. closure)from week 1 (˜2 cm long diameter) through to week 6 (˜0.75 cm long axisdiameter). In the column of insets b-j), the real-time fluorescenceimaging of endogenous bacterial fluorescence (autofluorescence) in andaround the wound can be tracked over time, and correlated with the whitelight images and wound closure measurements (column of insets a-i).Inset b) shows a distinct green band of fluorescence at the immediateboundary of the wound (yellow arrow; shown to be contaminated heavygrowth of Staphylococcus aureus), and this band changes over time as thewound heals. Red fluorescence bacteria are also seen further away fromthe wound (orange arrow), and their biodistribution changes over time(see column of insets b-j). The wound-to-periwound-to-normal tissueboundaries can be seen clearly by fluorescence in image inset j).Connective tissue (in this example, collagens) in normal skin appear aspale green fluorescence (inset j) and connective tissue remodelingduring wound healing can be monitored over time, during various woundtreatments including, as is the case here, hyperbaric oxygen therapy ofchronic wounds.

FIG. 21 illustrates use of the imaging device for targeting bacterialswabs during routine wound assessment in the clinic. Under fluorescenceimaging, the swab can be directed or targeted to specific areas ofbacterial contamination/infection using fluorescence image-guidance inreal-time. This may decrease the potential for contamination ofnon-infected tissues by reducing the spread of bacteria during routineswabbing procedures, which may be a problem in conventional woundswabbing methods. Swab results from this sample were determined to beStaphylococcus aureus (with few Gram positive bacilli and rare Grampositive cocci, confirmed by microscopy).

FIG. 22 shows an example of the co-registration of a) white light and b)corresponding fluorescence images made with the imaging device in apatient with diabetes-associated non-healing foot ulcers. Using anon-contact temperature measuring probe (inset in a) with cross-lasersighting, direct temperature measurements were made on normal skin(yellow “3 and 4”) and within the foot ulcers (yellow “1 and 2”)(infected with Pseudomonas aeruginosa, as confirmed by bacteriologicalculture), indicating the ability to add temperature-based information tothe wound assessment during the clinical examination. Infected woundshave elevated temperatures, as seen by the average 34.45° C. in theinfected wounds compared with the 30.75° C. on the normal skin surface,and these data illustrate the possibility of multimodality measurementswhich include white light, fluorescence and thermal information forwound health/infectious assessment in real-time. Note that bothnon-healing wounds on this patient's right foot contained heavy growthof Pseudomonas aeruginosa (in addition to Gram positive cocci and Gramnegative bacilli), which in this example appear as bright greenfluorescent areas within the wound (inset b).

FIG. 23 shows an example of the use of the imaging device for monitoringa pressure ulcer. Inset a) White light image taken with the imagingdevice of the right foot of a Caucasian diabetic patient with a pressureulcer is shown. Inset b) Corresponding fluorescence image shows thebright red fluorescence of bacteria (bacteriology results confirmedpresence of heavy growth of Staphylococcus aureus) which are invisibleunder standard white light examination (yellow arrows). Dead skinappears as a white/pale light green color (white arrows). Note the heavygrowth of Staphylococcus aureus bacteria around the periphery of thenon-healing open wounds (yellow arrows). Inset c) Shows the fluorescenceimaging of a topically applied silver antimicrobial dressing. Theimaging device may be used to detect the endogenous fluorescence signalfrom advanced wound care products (e.g., hydrogels, wound dressings,etc.) or the fluorescence signals from such products which have beenprepared with a fluorescent dye with an emission wavelength within thedetection sensitivity of the imaging detector on the device. The devicemay be used for image-guided delivery/application of advanced wound caretreatment products and to subsequently monitor their distribution andclearance over time.

FIG. 24 shows an example of the use of the device for monitoring apressure ulcer. Inset a) White light image taken with the device of theright foot of a Caucasian diabetic patient with a pressure ulcer. Insetb) Corresponding fluorescence image shows the bright red fluorescentarea of bacteria (bacteriology results confirmed presence of heavygrowth of Staphylococcus aureus, SA) at the wound edge and bright greenfluorescent bacteria (bacteriology results confirmed presence of heavygrowth of Pseudomonas aeruginosa, PA) which are both invisible understandard white light examination. Inset c) Fluorescence spectroscopytaken of the wound revealed unique spectral differences between thesetwo bacterial species: SA has a characteristic red (about 630 nm)autofluorescence emission peak, while PA lacks the red fluorescence buthas a strong green autofluorescence peak at around 480 nm.

The handheld device spectrally distinguishes bacteria from connectivetissues and blood in vivo. Using λexc=405_20 nm and λemiss=500 to 550nm, 590 to 690 nm, the device detects AF signals of S. aureus,Staphylococcus epidermidis, P. aeruginosa, Candida, Serratia marcescens,Viridans streptococci (α-hemolytic streptococci), Streptococcus pyogenes(β-hemolytic streptococci), Corynebacterium diphtheriae, Enterobacter,Enterococcus, and methicillin-resistant S. aureus (MRSA), as verified bymicrobiological swab cultures (data from a human clinical trial by ourgroup to be published in a forthcoming paper). This is a representativeof the major types of pathogenic bacteria commonly found in infectedwounds. Clinical microbiology tests confirmed that S. aureus, S.epidermidis, Candida, S. marcescens, Viridans streptococci,Corynebacterium diphtheriae, S. pyogenes, Enterobacter, and Enterococcusproduced red FL (from porphyrin) while P. aeruginosa produced abluish-green FL (from pyoverdin) detected by the handheld device. Thesespectral characteristics differ significantly from connective tissues(collagen, elastin) and blood, which appear green and dark red,respectively. A representative image of these spectral characteristicsis shown in FIG. 24.

FIG. 25 shows an example of the use of the device for monitoring achronic non-healing wound. Inset a) White light image taken with theimaging device of chronic non-healing wounds in 44 year old black malepatient with Type II diabetes is shown. Bacteria cannot be visualized bystandard white light visualization (see column of insets a-g) used inconventional clinical examination of the wounds. Column of insets b-h)Corresponding fluorescence image of the same wounds (405 nm excitation,500-550 nm emission (green), >600 nm emission (red)). This patientpresented with multiple open non-healing wounds. Swab cultures takenfrom each wound area using the fluorescence image-guidance revealed theheavy growths of Pseudomonas aruginosa (yellow arrow) which appearbright green fluorescent, and Serratia marcescens (circles) which appearred fluorescent. (Scale bar in cm).

FIG. 26 is a schematic diagram illustrating an example of a use of“calibration” targets, which may be custom-designed, multi-purpose,and/or disposable, for use during wound imaging with the imaging device.The strip, which in this example is adhesive, may contain a combinationof one or more of: spatial measurement tools (e.g., length scale),information barcode for integrating patient-specific medicalinformation, and impregnated concentration-gradients of fluorescent dyesfor real-time fluorescence image calibration during imaging. For thelatter, multiple concentrations of various exogenous fluorescent dyes orother fluorescence agents (e.g., quantum dots) may be used formultiplexed fluorescence intensity calibration, for example when morethan one exogenous fluorescently-labeled probe is used fortissue/cell/molecular-targeted molecular imaging of wounds in vivo.

FIG. 27 shows an example of the use of an embodiment of the imagingdevice for monitoring bacteria, for example for monitoring a treatmentresponse. Inset a) Fluorescence microscopy image of a live/dead bacteriastain sold by Invitrogen Corp. (i.e., BacLight product). Inset b)Fluorescence microscopy image of a Gram staining bacteria labeling stainsold by Invitrogen Corp. Using the imaging device inset (c) with suchproducts, live (green) and dead (red) bacteria inset (e) may bedistinguished in real-time ex vivo (e.g., on the swab or tissue biopsy)following bacterial swabbing of a wound, or other body surface, forexample, in the swabbing of the oral buccal cheek, as in inset d). Thisreal-time bacterial Gram staining or live/dead image-based assessmentmay be useful for real-time or relatively rapid bacteriology resultsthat may be used for refining treatments, such as antibiotic or otherdisinfective treatments, or for monitoring treatment response.

FIG. 28 shows an example of the use of the device used for imaging oftoe nail infection. Inset a) White light and inset b) correspondingautofluorescence of the right toe of a subject demonstrating theenhanced contrast of the infection that fluorescence imaging providescompared to white light visualization (405 nm excitation, 500-550 nmemission (green), >600 nm emission (red)).

Examples

FIG. 29 shows an example of the device being used for non-invasiveautofluorescence detection of collagen and varies bacterial species onthe skin surface of a pig meat sample. In contrast to white lightimaging, autofluorescence imaging was able to detect the presence ofseveral bacterial species 24 h after they were topically applied tosmall incisions made in the skin (i.e., streptococcus pyogenes, serratiamarcescens, staphylococcus aureus, staphylococcus epidermidis,escherichia coli, and pseudomonas aeruginosa). Inset a) shows whitelight images of pig meat used for testing. Several bacterial specieswere applied to small incisions made in the skin at Day 0, and werelabelled as follows: 1) streptococcus pyogenes, 2) serratia marcescens,3) staphylococcus aureus, 4) staphylococcus epidermidis, 5) escherichiacoli, and 6) pseudomonas aeruginosa. The imaging device was used todetect collagen and bacterial autofluorescence over time. Connectivetissue fluorescence was intense and easily detected as well. Somebacterial species (e.g., pseudomonas aeruginosa) produces significantgreen autofluorescence (450-505 nm) which saturated the device's camera.Inset b) shows autofluorescence image at Day 0, magnified in inset c).

The device was also able to detect spreading of the bacteria over thesurface of the meat over time. Inset d) shows an image at Day 1, andinset f) shows an image at Day 3, as the meat sample was maintained at37° C. Red fluorescence can be seen in some of the wound sites (5, 6) ininset c). As shown in inset d) and magnified in inset e), after 24 h,the device detects a dramatic increase in bacterial autofluorescencefrom wound site 5) escherichia coli and 6) pseudomonas aeruginosa, withthe latter producing significant green and red autofluorescence. Insetsc) and e) show the device detecting fluorescence using a dual band(450-505 nm green and 590-650 nm) on the left and a single band filter(635+/−10 nm) on the right, of the wound surface. As shown in inset f),by Day 3, the device detects the significant increase in bacterialautofluorescence (in green and red) from the other wound sites, as wellas the bacterial contamination (indicated by the arrow in inset f) onthe styrofoam container in which the meat sample was kept. The devicewas also able to detect spreading of the bacteria over the surface ofthe meat. This demonstrates the real-time detection of bacterial specieson simulated wounds, the growth of those bacteria over time, and thecapability of the device to provide longitudinal monitoring of bacterialgrowth in wounds. The device may provide critical information on thebiodistribution of the bacteria on the wound surface which may be usefulfor targeting bacterial swabbing and tissue biopsies. Note, in insets d)and f), the intense green fluorescence signal from endogenous collagenat the edge of the pig meat sample.

This example demonstrates the use of the device for real-time detectionof biological changes in connective tissue and bacterial growth based onautofluorescence alone, suggesting a practical capability of the deviceto provide longitudinal monitoring of bacterial growth in wounds.

Referring again to FIG. 3, the images show examples of the device usedfor autofluorescence detection of connective tissues (e.g., collagen,elastin) and bacteria on the muscle surface of a pig meat sample. Inseta) shows that white light image of pig meat used for testing shows noobvious signs of bacterial/microbial contamination or spoilage. However,as seen in inset b), imaging of the same area with the device underblue/violet light excitation revealed a bright red fluorescent area ofthe muscle indicating the potential for bacterial contamination comparedwith the adjacent side of muscle. Extremely bright greenautofluorescence of collagen can also be seen at the edge of the skin.In inset c), the device was used to surgically interrogate suspiciousred fluorescence further to provide a targeted biopsy for subsequentpathology or bacteriology. Note also the capability of the device todetect by fluorescence the contamination (arrow) of the surgicalinstrument (e.g., forceps) during surgery. In inset d), the device wasused to target the collection of fluorescence spectroscopy using a fibreoptic probe of an area suspected to be infected by bacteria (inset showsthe device being used to target the spectroscopy probe in the same areaof red fluorescent muscle in insets b), c). e) show an example of thedevice being used to detect contamination by various thin films ofbacteria on the surface of the Styrofoam container on which the meatsample was kept. Autofluorescence of the bacteria appears as streaks ofgreen and red fluorescence under violet/blue excitation light from thevarious bacterial species previously applied to the meat. Thus, thedevice is capable of detecting bacteria on non-biological surfaces wherethey are occult under standard white light viewing (as in inset a).

In addition to detection of bacteria in wounds and on the skin surface,the device was also able to identify suspicious areas of muscle tissue,which may then be interrogated further by surgery or targeted biopsy forpathological verification, or by other optical means such asfluorescence spectroscopy using a fiber optic probe. Also, it detectedcontamination by various bacteria on the surface of the Styrofoamcontainer on which the meat sample was kept. Autofluorescence of thebacteria appears as streaks of green and red fluorescence underviolet/blue excitation light from the various bacterial speciespreviously applied to the meat.

In order to determine the autofluorescence characteristics of bacteriagrowing in culture and in the simulated skin wounds,hyperspectral/multispectral fluorescence imaging was used toquantitatively measure the fluorescence intensity spectra from thebacteria under violet/blue light excitation. Reference is now made toFIG. 30. In FIG. 30, the device was used to detect fluorescence frombacteria growing in agar plates and on the surface of a simulated woundon pig meat, as discussed above for FIGS. 12 and 29. Bacterialautofluorescence was detected in the green and red wavelength rangesusing the device in the culture inset (a) and meat samples inset (d).Hyperspectral/multispectral imaging was used to image the bacteria (E.Coli) in culture inset (b) and to measure the quantitative fluorescenceintensity spectra from the bacteria (red line—porphyrins, greencytoplasm, blue—agar background) inset (c). The red arrow shows the 635nm peak of porphyrin fluorescence detected in the bacteria.Hyperspectral/multispectral imaging also confirmed the strong greenfluorescence (*, right square in inset d) from P. aruginosa (with littleporphyrin fluorescence, yellow line in inset f) compared to E. coli(left square in inset d) where significant porphyrin red fluorescencewas detected. Insets e) and g) show the color-codedhyperspectral/multispectral images corresponding to P. aeruginosa and E.coli, respectively, from the meat surface after 2 days of growth(incubated at 37° C.); and insets f) and h) show the correspondingcolor-coded fluorescence spectroscopy. In inset i), excitation-emissionmatrices (EEM) were also measured for the various bacterial species insolution, demonstrating the ability to select the optimum excitation andemission wavelength bandwidths for use with optical filters in theimaging device. The EEM for E. coli shows strong green fluorescence aswell as significant red fluorescence from endogenous bacterialporphyrins (arrow).

This example shows that bacteria emit green and red autofluorescence,with some species (e.g., pseudomonas aeruginosa) producing more of theformer. Escherichia coli produced significant red autofluorescence fromendogenous porphyrins. Such intrinsic spectral differences betweenbacterial species are significant because it may provide a means ofdifferentiating between different bacterial species usingautofluorescence alone. Excitation-emission matrices (EEMs) were alsomeasured for each of the bacterial species used in these pilot studies,which confirmed that under violet/blue light excitation, all speciesproduced significant green and/or red fluorescence, the latter beingproduced by porphyrins. Spectral information derived fromexcitation-emission matrices may aid in optimizing the selection ofexcitation and emission wavelength bandwidths for use with opticalfilters in the imaging device to permit inter-bacterial speciesdifferentiating ex vivo and in vivo. In this way, the device may be usedto detect subtle changes in the presence and amount of endogenousconnective tissues (e.g. collagens and elastins) as well as bacteriaand/or other microorganisms, such as yeast, fungus and mold withinwounds and surrounding normal tissues, based on unique autofluorescencesignatures of these biological components.

This device may be used as an imaging and/or monitoring device inclinical microbiology laboratories. For example, the device may be usedfor quantitative imaging of bacterial colonies and quantifying colonygrowth in common microbiology assays. Fluorescence imaging of bacterialcolonies may be used to determine growth kinetics.Imaging of Blood in Wounds

Angiogenesis, the growth of new blood vessels, is an important naturalprocess required for healing wounds and for restoring blood flow totissues after injury or insult. Angiogenesis therapies, which aredesigned to “turn on” new capillary growth, are revolutionizing medicineby providing a unified approach for treating crippling andlife-threatening conditions. Angiogenesis is a physiological processrequired for wound healing. Immediately following injury, angiogenesisis initiated by multiple molecular signals, including hemostaticfactors, inflammation, cytokine growth factors, and cell-matrixinteractions. New capillaries proliferate via a cascade of biologicalevents to form granulation tissue in the wound bed. This process may besustained until the terminal stages of healing, when angiogenesis ishalted by diminished levels of growth factors, resolution ofinflammation, stabilized tissue matrix, and endogenous inhibitors ofangiogenesis. Defects in the angiogenesis pathway impair granulation anddelay healing, and these are evident in chronic wounds. By illuminatingthe tissue surface with selected narrow wavelength bands (e.g., blue,green and red components) of light or detecting the reflectance of whitelight within several narrow bandwidths of the visible spectrum (e.g.,selected wavelengths of peak absorption from the blood absorptionspectrum of white light), the device may also be used to image thepresence of blood and microvascular networks within and around thewound, including the surrounding normal tissue, thus also revealingareas of erythema and inflammation.

Reference is now made to FIG. 31. The device may use individual opticalfilters (e.g., 405 nm, 546 nm, 600 nm, +/−25 nm each) in order todemonstrate the possibility of imaging blood and microvasculature inwounds. White light images of a wound may be collected with the deviceand then the device, equipped with a triple band-pass filter (e.g., 405nm, 546 nm, 600 nm, +/−25 nm each), placed in front of the imagingdetector may image the separate narrow bandwidths of blue (B), green(G), and red (R) reflected light components from the wound. Thesewavelength bands may be selected based on the peak absorptionwavelengths of blood, containing both oxygenated and deoxygenatedhemoglobin, in the visible light wavelength range. The resulting imagesmay yield the relative absorption, and thus reflectance, of visiblelight by blood in the field of view. The resulting ‘blood absorption’image yields a high contrast image of the presence of blood and/ormicrovascular networks in the wound and surrounding normal tissues. Theclinician may select the appropriate optical filter set for use with thedevice to obtain images of blood and/or microvascular distributionwithin the wound and the combine this information with one or both ofautofluorescence imaging and imaging with exogenous contrast agents.This may provide a comprehensive information set of the wound andsurrounding normal tissues at the morphological, topographical,anatomical, physiological, biological and molecular levels, whichcurrently may not be possible within conventional wound care practice.

FIG. 31 shows examples of the device used for imaging of blood andmicrovasculature in wounds. The device was used to image a piece offilter paper stained with blood inset (a) and the ear of a mouse duringsurgery inset (b). White light images were collected of each specimenusing the imaging device, in non-fluorescence mode, and then the devicewas equipped with a triple band-pass filter placed in front of theimaging detector (405 nm, 546 nm, 600 nm, +/−25 nm each) to image theseparate narrow bandwidths of blue (B), green (G), and red (R) reflectedlight components from the specimens. These wavelength bands wereselected based on the peak absorption wavelengths of blood in thevisible light wavelength range (inset in a) shows the absorptionspectral profile for oxy- and deoxygenated hemoglobin in blood. Thisshows that using a simple multiband transmission filter, it is possibleto combine the three B, G, R images into a single ‘white lightequivalent’ image that measures the relative absorption of light byblood in the field of view. The resulting ‘blood absorption’ imageyields a high contrast image of the presence of blood containing bothoxy- and deoxygenated hemoglobin. The device may be used with narrowerbandwidth filters to yield higher contrast images of blood absorption inwounds, for example.

The regulation of angiogenesis over time during wound repair in vivo hasbeen largely unexplored, due to difficulties in observing events withinblood vessels. Although initial tests of the imaging device wereexploratory, simple modification of the existing prototype device mayallow longitudinal imaging of dynamic changes in blood supply andmicrovascular networks during the wound healing process in vivo.

In general, the device may be used to image and/or monitor targets suchas a skin target, an oral target, an ear-nose-throat target, an oculartarget, a genital target, an anal target, and any other suitable targetson a subject.

Use in Clinical Care

Although current wound management practice aims to decrease themorbidity and mortality of wounds in patients, a limitation is theavailability of health care resources. The potential of incorporatingthe technology of telemedicine into wound care needs is currently beingexplored. Wound care is a representation of the care of chronic anddebilitating conditions that require long-term specialized care. Themajor effect of improved living conditions and advances in health careglobally has led to people living longer. Therefore, the percentage ofworlds' elderly and those with chronic medical conditions that wouldrequire medical attention is rising. With the escalating costs of healthcare, and the push of the industry towards outpatient care, this is apart of the health care crisis that is demanding immediate attention.

The present device may provide biologically-relevant information aboutwounds and may exploit the emerging telemedicine (e.g., E-health)infrastructure to provide a solution for mobile wound care technologyand may greatly impact wound health care treatment. Wound care accountsfor a large percentage of home visits conducted by nurses and healthcare workers. Despite best practices some wounds do not heal as expectedand require the services of a clinical specialist. The device describedhere may enable access to specialized clinical resources to help treatwounds from the convenience of the patient's home or chronic carefacility, which decreases travel time for clients, increasesavailability to clinical wound specialists, and may reduce costs to thehealth care system.

Different uses of the imaging device have been discussed for woundassessment, monitoring and care management. The device may be used todetect and monitor changes in connective tissues (e.g., collagen,elastin) and blood/vascular supply during the wound healing process,monitor tissue necrosis and exudate in wounds based on fluorescence,detect and diagnose wound infections including potentially indicatingcritical ‘clinically significant’ categories of the presence of bacteriaor micro-organisms (e.g., for detecting contamination, colonization,critical colonization and infection) at the surface and deep withinwounds, provide topographic information of the wound, and identify woundmargins and surrounding normal tissues. Tissue fluorescence andreflectance imaging data may be ‘mapped’ onto the white light images ofthe wound thereby permitting visualization within the wound and thesurrounding normal tissues of essential wound biochemical andphotobiological (e.g., fluorescence) information, which has not beenpossible to date. Real-time imaging of wounds may be performed over timeto monitoring changes in wound healing, and to potentially monitor theeffectiveness of treatments by providing useful information aboutunderlying biological changes that are occurring at the tissue/cellularlevel (e.g., matrix remodeling, inflammation, infection and necrosis).This may provide quantitative and objective wound information fordetection, diagnosis and treatment monitoring in patients. Inparticular, the device may be used to monitor and/or track theeffectiveness of therapy at a biological level (e.g., on a bacteriallevel), which may provide more information than monitoring only themacroscopic/morphological appearance using white light.

The device may provide real-time non-invasive image-guided biopsytargeting, clinical procedural guidance, tissue characterization, andmay enable image-guided treatment using conventional and emergingmodalities (e.g., PDT). In addition, use of the imaging device may beused to correlate critical biological and molecular wound informationobtained by fluorescence (e.g., endogenous tissue autofluorescenceand/or administration of exogenous molecular-biomarker targetedfluorescence contrast agents) with existing and emerging clinical woundcare assessment and treatment guides, such as the NERDS and STONESguidelines proposed by Sibbald et al. (Sibbald et al. IncreasedBacterial Burden and Infection: The Story of NERDS and STONES. ADV SKINWOUND CARE 2006; 19:447-61). The fluorescence imaging data obtained withthe device may be used to characterize, spatially and spectrally,bacterial balance and burden at the superficial and deep levels ofwounds. The device may provide real-time non-invasive image-guidedbiopsy targeting, clinical procedural guidance, tissue characterization,and may enable image-guided treatment using conventional and emergingtreatment modalities (e.g., photodynamic therapy, PDT). The device maybe used within the clinical setting and integrated into conventionalclinical wound care regimens, and may have a distinct role in areas ofinfectious diseases. It should be noted as well that this device mayalso be used for real-time analysis, monitoring and care for chronic andacute wounds in animals and pets, via conventional veterinary care.

This device may allow real-time wound healing assessment for a largepatient cohort base. In particular, elderly people, diabetics,immuno-suppressed and immobilized individuals have an increasedincidence of chronic wounds and other dermal afflictions that resultfrom poor circulation and immobility, e.g. pressure ulcers such as bedsores, venous stasis ulcers, and diabetic ulcers. These chronicconditions greatly increase the cost of care and reduce the patient'squality of life. As these groups are growing in number, the need foradvanced wound care products will increase. This device may impactpatient care by allowing a cost-effective means of monitoring chronicand acute wounds in a number of settings, including hospitals,ambulatory clinics, chronic care facilities, in-home-visit health care,emergency rooms and other critical areas in health care facilities.Further, such a ‘hand-held’ and portable imaging device may be easilycarried and used by nursing and ambulance staff. Early identification ofscarring, which is related to connective tissue production andre-modeling of the wound, and bacterial infections may be detected andtreated appropriately, something that is currently difficult. Inaddition, recent developments in advanced wound-care products includingmultiple dressing types (e.g., film, hydrocolloid, foam, anti-microbial,alginate, non-adherent, impregnated), hydrogels, wound cleansers anddebriding agents, tissue engineered products (e.g., skin replacements,substitutes, and tissue-engineered products such as syntheticpolymer-based biological tissue and growth factors), wound cleansers,pharmacological products, and physical therapies may also benefit fromthe device developed here as it may allow image-based longitudinalmonitoring of the effectiveness of such treatments. Physical therapiesmay include hydrotherapy, electrical stimulation, electromagneticstimulation devices, ultraviolet therapy, hyperbaric oxygen therapy,ultrasound devices, laser/light emitting diode (LED) devices, and woundimaging/documentation. Additional therapies may include, for example,antibiotics, wound debridement, application of wound dressings, andwound cleaning.

Wound tissue analysis is typically required for the assessment of thehealing of skin wounds. Percentage of the granulation tissue, fibrin andnecrosis in the wound, and their change during treatment may provideuseful information that may guide wound treatment. Image analysis mayinclude advanced statistical pattern recognition and classificationalgorithms to identify individual pixels within the fluorescence woundimages collected with the device based on the optical information of thewound and surrounding normal tissue. Thus, image analysis may allowwound images to be mapped into various components of the wound,including total wound area, epithelialization, granulation, slough,necrotic, hypergranulation, infected, undermining, and surroundingtissue margins. This has an added advantage of providing relativelyrapid determination of wound healing rates, as well as informing guidepatient management decisions.

FIG. 32 illustrates the projected management workflow for the imagingdevice in a clinical wound care setting. The device may be easilyintegrated into routine wound assessment, diagnosis, treatment andlongitudinal monitoring of response, and may provide critical biologicaland molecular information of the wound in real-time for rapiddecision-making during adaptive interventions.

This device may be easily integrated into existing health-care computerinfrastructures (e.g., desktop and pocket PCs used by a growing numberof physicians or other health care professionals) for longitudinal imagecataloguing for patient wound management within the conventionalclinical environment. The wireless receiving and transmission of datacapabilities of the device may allow monitoring of wound care andhealing remotely through existing and future wireless telemedicineinfrastructure. The device may be used to transfer essential medicaldata (e.g., wound health status) via the internet or over wirelessservices, such as cellular telephone, PDA or Smartphone services, toremote sites which may permit remote medical interventions, with afurther utility in military medical applications for battlefield woundmanagement. The device may allow real-time surface imaging of woundsites and may be easily carried by point-of-care personnel in clinicalsettings. Using cost-effective highly sensitive commercially availabledigital imaging devices, such as digital cameras, cellular phones, PDAs,laptop computers, tablet PCs, webcams, and Smart phones, etc. as theimage capture or recording component, the device may offer image-baseddocumentation of wound healing and tracking of treatment effectiveness.Also, this technology may be adapted to also function in ‘wireless’ modeto permit remote medical interventions by potentially adapting it foruse with high-resolution digital cameras embedded incommercially-available cellular telephones.

By using web-based telemedicine and remote medical monitoringinfrastructure, the imaging device may be integrated into a‘store-and-forward’ concept of wound assessment systems. In addition toproviding digital images, such a system may present a comprehensive setof clinical data that meet the recommendations of clinical practiceguidelines. The presently-disclosed device may integrate into acomputer-based wound assessment system (e.g., with image analysissoftware) to be used by a health care facility to enhance existingclinical databases and support the implementation of evidence—basedpractice guidelines. Such an integrated telemedicine infrastructure maybe used for monitoring patients at home or in long-term-care facilities,who may benefit from routine monitoring by qualified clinicians butcurrently do not have access to this care. This device may be furtherdeveloped into a portable handheld point-of-care diagnostic system,which may represent a major advance in detecting, monitoring, treating,and preventing infectious disease spread in the developed and developingworlds. This knowledge may significantly improve the diagnostic toolsavailable to practitioners who treat chronic wounds in settings wherequantitative cultures are inaccessible.

The device may allow digital imaging with optical and digital zoomingcapabilities (e.g., those embedded in commonly available digital imagingdevices). Still or video image quality may be in ‘high-definition’format to achieve high spatial resolution imaging of the tissue surface.Images may be recorded as still/freeze frame and/or in video/movieformat and printed using standard imaging printing protocols which do(e.g., connected via USB) or do not (e.g., PictBridge) require apersonal computer. The images/video data may be transferred to apersonal computer for data archival storage and/or image viewing and/oranalysis/manipulation. The device may also transfer data to a printer orpersonal computer using wired or wireless capabilities (e.g.,Bluetooth). Visualization may be performed on the hand-held devicescreen and/or in addition to simultaneous viewing on a videoscreen/monitor (e.g., head-mounted displays and glasses) using standardoutput video cables. This device may display, in combination orseparately, optical wavelength and fluorescence/reflectance intensityinformation with spatial dimensions of the imaged scene to allowquantitative measurements of distances (e.g., monitoring changes tissuemorphology/topography) over time. The device may also allow digitalimage/video storage/cataloguing of images and related patient medicaldata, for example using dedicated software with imaging analysiscapabilities and/or diagnostic algorithms.

Image Analysis

Image analysis may be used together with the device to quantitativelymeasure fluorescence intensities and relative changes in multiplefluorescence spectra (e.g., multiplexed imaging) of the exogenousoptical molecular targeting probes in the wound and surrounding normaltissues. The biodistributions of the fluorescent probes may bedetermined based on the fluorescence images collected and these may bemonitored over time between individual clinical wound imaging sessionsfor change. By determining the presence and relative changes inabundance quantitatively, using the device, of each and all of thespectrally-unique fluorescent probes, the clinical operator maydetermine in real-time or near real-time the health and/or healingstatus and response to treatment over time of a given wound, for exampleby using a look-up table in which specific tissue, cellular andmolecular signals are displayed in correlation to wound health, healingand response status, an example of which is shown in FIG. 33. This maypermit the clinician to determine whether a wound is healing based onbiological and molecular information which may not be possible otherwisewith existing technologies. Furthermore, the presence and abundance ofbacteria/microorganisms and their response to treatment may offer ameans to adapt the therapy in real-time instead of incurring delays inresponse assessment with conventional bacteriological testing of woundcultures.

Image analysis techniques may be used to calibrate the initial or firstimages of the wound using a portable fluorescent standard placed withinthe field of view during imaging with the device. The image analysis mayalso permit false or pseudo color display on a monitor fordifferentiating different biological (e.g., tissue, cellular, andmolecular) components of the wound and surrounding normal tissuesincluding those biomarkers identified by autofluorescence and thoseidentified by the use of exogenous targeted or untargetedfluorescence/absorption contrast agents.

Examples of such biomarkers are listed in FIG. 34 and illustrated inFIG. 35. In FIG. 35, the diagram shows mechanisms of wound healing inhealthy people versus people with diabetic wounds. In healthyindividuals (left), the acute wound healing process is guided andmaintained through integration of multiple molecular signals (e.g., inthe form of cytokines and chemokines) released by keratinocytes,fibroblasts, endothelial cells, macrophages, and platelets. Duringwound-induced hypoxia, vascular endothelial growth factor (VEGF)released by macrophages, fibroblasts, and epithelial cells induces thephosphorylation and activation of eNOS in the bone marrow, resulting inan increase in NO levels, which triggers the mobilization of bone marrowEPCs to the circulation. For example, the chemokine SDF-1α promotes thehoming of these EPCs to the site of injury, where they participate inneovasculogenesis. In a murine model of diabetes (right), eNOSphosphorylation in the bone marrow is impaired, which directly limitsEPC mobilization from the bone marrow into the circulation. SDF-1αexpression is decreased in epithelial cells and myofibroblasts in thediabetic wound, which prevents EPC homing to wounds and therefore limitswound healing. It has been shown that establishing hyperoxia in woundtissue (e.g., via HBO therapy) activated many NOS isoforms, increased NOlevels, and enhanced EPC mobilization to the circulation. However, localadministration of SDF-1α was required to trigger homing of these cellsto the wound site. These results suggest that HBO therapy combined withSDF-1α administration may be a potential therapeutic option toaccelerate diabetic wound healing alone or in combination with existingclinical protocols.

Pre-assigned color maps may be used to display simultaneously thebiological components of the wound and surrounding normal tissuesincluding connective tissues, blood, microvascularity, bacteria,microorganisms, etc. as well as fluorescently labeleddrugs/pharmacological agents. This may permit visualization in real-timeor near real-time (e.g., less than 1 minute) of the health, healing andinfectious status of the wound area.

The image analysis algorithms may provide one or more of the followingfeatures:

Patient Digital Image Management

-   -   Integration of a variety of image acquisition devices    -   Records all imaging parameters including all exogenous        fluorescence contrast agents    -   Multiple scale and calibrations settings    -   Built-in spectral image un-mixing and calculation algorithms for        quantitative determination of tissue/bacterial autofluorescence        and exogenous agent fluorescence signals    -   Convenient annotation tools    -   Digital archiving    -   Web publishing        Basic Image Processing and Analysis    -   Complete suite of image processing and quantitative analysis        functions Image stitching algorithms will allow stitching of a        series of panoramic or partially overlapping images of a wound        into a single image, either in automated or manual mode.    -   Easy to use measurement tools    -   Intuitive set up of processing parameters    -   Convenient manual editor        Report Generation    -   Powerful image report generator with professional templates        which may be integrated into existing clinical report        infrastructures, or telemedicine/e-health patient medical data        infrastructures. Reports may be exported to PDF, Word, Excel,        for example.        Large Library of Automated Solutions    -   Customized automated solutions for various areas of wound        assessment including quantitative image analysis.

Although image analysis algorithm, techniques, or software have beendescribed, this description also extends to a computing device, asystem, and a method for carrying out this image analysis.

Image-guidance

The device may also be useful for providing fluorescent image-guidance,for example in surgical procedures, even without the use of dyes ormarkers. Certain tissues and/or organs may have different fluorescentspectra (e.g., endogenous fluorescence) when viewed using the imagingdevice, or example under certain excitation light conditions.

FIG. 36 demonstrates the usefulness of the device for fluorescenceimaging-assisted surgery. With the aid of fluorescence imaging using thedevice, different organs of a mouse model may be more clearlydistinguishable than under white light. Insets b, c and g show the mousemodel under white light. Insets a, d-f and h-j show the mouse model asimaged with the device.

FIG. 37 shows an example of the use of the device for imaging smallanimal models. Here, the mouse dorsal skin-fold window chamber is imagedunder white light (insets a, c) and fluorescence (insets b, d). Note thehigh-resolution white light and fluorescence images obtained by thedevice. The feet and face appear bright red fluorescent due toendogenous autofluorescence from the cage bedding and food dustmaterials. (405 nm excitation; 490-550 nm and >600 nm emissionchannels).

Bioengineered Skin

Several bioengineered skin products or skin equivalents have becomeavailable commercially for the treatment of acute and chronic wounds, aswell as burn wounds. These have been developed and tested in humanwounds. Skin equivalents may contain living cells, such as fibroblastsor keratinocytes, or both, while others are made of acellular materialsor extracts of living cells. The clinical effect of these constructs is15-20% better than conventional ‘control’ therapy, but there is debateover what constitutes an appropriate control. Bioengineered skin maywork by delivering living cells which are known as a ‘smart material’because they are capable of adapting to their environment. There isevidence that some of these living constructs are able to release growthfactors and cytokines. Exogenous fluorescent molecular agents may beused in conjunction with such skin substitutes to determine completenessof engraftment as well as biological response of the wound to thetherapy. The healing of full-thickness skin defects may requireextensive synthesis and remodeling of dermal and epidermal components.Fibroblasts play an important role in this process and are beingincorporated in the latest generation of artificial dermal substitutes.

The imaging device described here may be used to determine the fate offibroblasts seeded in skin substitute and the influence of the seededfibroblasts on cell migration and dermal substitute degradation aftertransplantation to wound site can be determined. Wounds may be treatedwith either dermal substitutes seeded with autologous fibroblasts oracellular substitutes. Seeded fibroblasts, labeled with a fluorescentcell marker, may then be detected in the wounds with fluorescenceimaging device and then quantitatively assessed using image analysis,for example as described above.

Polymer-Based Therapeutic Agents

There are a number of commercially available medical polymer productsmade for wound care. For example, Rimon Therapeutics produces Theramers™(www.rimontherapeutics.com) which are medical polymers that havebiological activity in and of themselves, without the use of drugs.Rimon Therapeutics produces the following wound care products, which canbe made to be uniquely fluorescent, when excited by 405 nm excitationlight: Angiogenic Theramer™, which induces new blood vessel development(i.e., angiogenesis) in wounds or other ischemic tissue; MI Theramer™,which inhibits the activity of matrix metalloproteases (MMPs), aubiquitous group of enzymes that are implicated in many conditions inwhich tissue is weakened or destroyed; AM Theramer™, a thermoplasticthat kills gram positive and gram negative bacteria without harmingmammalian cells; and ThermaGel™, a polymer that changes from a liquid toa strong gel reversibly around body temperature. These can each be madeto be fluorescent by addition of fluorescent dyes or fluorescentnanoparticles selected to be excited, for example, at 405 nm light withlonger wavelength fluorescence emission.

By using the imaging device, the application of such fluorescent polymeragents may be guided by fluorescent imaging in real-time. This maypermit the Theramer agent to be accurately delivered/applied (e.g.,topically) to the wound site. Following application of the agent to thewound, the fluorescent imaging device may then be used to quantitativelydetermine the therapeutic effects of the Theramers on the wound as wellas track the biodistribution of these in the wound over time, in vivoand non-invasively. It may also be possible to add a molecular beacon,possibly having another fluorescent emission wavelength, to the MITheramer™ that can fluoresce in the presence of wound enzymes (e.g.,MMPs), and this may indicate in real-time the response of the wound tothe MI Theramer™. It may be possible to use one fluorescence emissionfor image-guided Theramer application to the wound site and anotherdifferent fluorescence emission for therapeutic response monitoring, andother fluorescence emissions for other measurements. The relativeeffectiveness of MMP inhibition and antimicrobial treatments may bedetermined simultaneously over time. Using image analysis, real-timecomparison of changes in fluorescence of these signals in the wound maybe possible. This adds a quantitative aspect to the device, and adds toits clinical usefulness.

It should be noted that other custom bio-safe fluorescence agents may beadded to the following materials which are currently used for woundcare. The fluorescent material may then be imaged and monitored usingthe device.

-   -   Moist Wound Dressings: This provides a moist conducive        environment for better healing rates as compared to traditional        dressings. The primary consumer base that manufacturers target        for these dressings is people over the age of 65 years,        suffering from chronic wounds such as pressure ulcers and venous        stasis ulcers. Those suffering from diabetes and as a result,        developed ulcers form a part of the target population.    -   Hydrogels: This adds moisture to dry wounds, creating a suitable        environment for faster healing. Their added feature is that they        may be used on infected wounds. These are also designed for dry        to lightly exudative wounds.    -   Hydrocolloid Dressings: Hydrocolloids seal the wound bed and        prevent loss of moisture. They form a gel upon absorbing        exudates to provide a moist healing environment. These are used        for light to moderately exudative wounds with no infection.    -   Alginate Dressings: These absorb wound exudates to form a gel        that provides a moist environment for healing. They are used        mainly for highly exudative wounds.    -   Foam Dressing: These absorb wound drainage and maintain a moist        wound surface, allowing an environment conducive for wound        healing. They are used on moderately exudative wounds.    -   Transparent Film Dressing: These are non-absorptive, but allow        moisture vapor permeability, thereby ensuring a moist wound        surface. They are intended for dry to lightly exudative wounds.        Examples include alginate foam transparent film dressings.    -   Antimicrobials: These provide antibacterial action to disinfect        the wound. Of particular interest is the use of nanocrystalline        silver dressings. The bio burden, particularly accumulated        proteases and toxins released by bacteria that hampers healing        and causes pain and exudation, is reduced significantly with the        extended release of silver.    -   Active Wound Dressings: These comprise highly evolved tissue        engineered products. Biomaterials and skin substitutes fall        under this category; these are composed entirely of biopolymers        such as hyaluronic acid and collagen or biopolymers in        conjunction with synthetic polymers like nylon. These dressings        actively promote wound healing by interacting either directly or        indirectly with the wound tissues. Skin substitutes are        bioengineered devices that impersonate the structure and        function of the skin.    -   Hyaluronic Acid: This is a natural component of the extra        cellular matrix, and plays a significant role in the formation        of granular tissue, re-epithelialization and remodeling. It        provides hydration to the skin and acts as an absorbent.

Other wound care products that may be imaged using the disclosed deviceinclude Theramers, silver-containing gels (e.g., hydrogels), artificialskin, ADD stem cells, anti-matrix metalloproteinases, and hyaluronicacid. Fluorescent agents may be added to other products to allow forimaging using the device. In some cases, the products may already beluminescent and may not require the addition of fluorescent agents.

The device may be used also to monitor the effects of such treatmentsover time.

Kits for Device

The imaging device may be provided in a kit, for example including thedevice and a fluorescing contrast agent. The contrast agent may be anyone or more of those described above. For example, the contrast agentmay be for labeling a biomarker in a wound, where the kit is for woundmonitoring applications.

FIG. 38 shows an example of a kit including the imaging device. Inset a)shows the handle and the touch-sensitive viewing screen, and inset b)shows external housing and excitation light sources. The imaging devicemay be used to scan the body surface of both human and veterinarypatients for image-based wound assessment, or for non-wound imagingapplications. The device and any accessories (e.g., electrical/batterypower supplies), potential exogenous fluorescence contrast agents, etc.)may be conveniently placed into hard-case containers for transportwithin clinical and non-clinical environments (including remote sites,home care and research laboratory settings).

The imaging device may be used in white light and fluorescence modes toimprove the administration of these treatments as well as monitor theireffectiveness over time non-invasively and quantitatively. The devicemay be used in combination with other imaging modalities, for examplethermal imaging methods, among others.

While the present disclosure has been disclosed in terms of exemplaryembodiments in order to facilitate better understanding of thedisclosure, it should be appreciated that the disclosure can be embodiedin various ways without departing from the principle of the disclosure.Therefore, the disclosure should be understood to include all possibleembodiments which can be embodied without departing from the principleof the disclosure set out in the appended claims. Furthermore, althoughthe present disclosure has been discussed with relation to woundimaging, monitoring, and analysis those of ordinary skill in the artwould understand that the present teachings as disclosed would workequally well in various other applications such as, for example,clinically- and research-based imaging of small and large (e.g.,veterinary) animals; detection and monitoring of contamination (e.g.,bacterial contamination) in food/animal product preparation in the meat,poultry, dairy, fish, agricultural industries; detection of ‘surfacecontamination’ (e.g., bacterial or biological contamination) in public(e.g., health care) and private settings; multi-spectral imaging anddetection of cancers in human and/or veterinary patients; as a researchtool for multi-spectral imaging and monitoring of cancers inexperimental animal models of human diseases (e.g., wound and cancers);forensic detection, for example of latent finger prints and biologicalfluids on non-biological surfaces; imaging and monitoring of dentalplaques, carries and cancers in the oral cavity; imaging and monitoringdevice in clinical microbiology laboratories; and testing anti-bacterial(e.g., antibiotic), disinfectant agents. The use of a fluorescentimaging device in such environments is disclosed in U.S. Pat. No.9,042,967 B2 to DaCosta et al., entitled “Device and Method for WoundImaging and Monitoring,” and issued on May 26, 2015, which isincorporated by reference herein. Additionally or alternatively, thedevice may be used for detecting and imaging of the presence of bacteriaor microbes and other pathogens on a variety of surfaces, materials,instruments (e.g., surgical instruments) in hospitals, chronic carefacilities, old age homes, and other health care settings wherecontamination may be the leading source of infection. The device may beused in conjunction with standard detection, identification andenumeration of indicator organisms and pathogens strategies.

For the purposes of this specification and appended claims, unlessotherwise indicated, all numbers expressing quantities, percentages orproportions, and other numerical values used in the specification andclaims, are to be understood as being modified in all instances by theterm “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the written description and claims areapproximations that may vary depending upon the desired propertiessought to be obtained by the present disclosure. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques.

It is noted that, as used in this specification and the appended claims,the singular forms “a,” “an,” and “the,” include plural referents unlessexpressly and unequivocally limited to one referent. Thus, for example,reference to “a sensor” includes two or more different sensors. As usedherein, the term “include” and its grammatical variants are intended tobe non-limiting, such that recitation of items in a list is not to theexclusion of other like items that can be substituted or added to thelisted items.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the system and method of thepresent disclosure without departing from the scope its teachings. Otherembodiments of the disclosure will be apparent to those skilled in theart from consideration of the specification and practice of theteachings disclosed herein. It is intended that the specification andembodiment described herein be considered as exemplary only.

The invention claimed is:
 1. A method of obtaining diagnostic dataregarding a target, comprising: directly illuminating at least a portionof a target with a uniform field of excitation light emitted by at leastone light source connected to a housing of a handheld device, the atleast one light source emitting at least one wavelength or wavelengthband causing at least one biomarker in the illuminated portion of thetarget to fluoresce; filtering optical signals responsive toillumination of the target with a filter of the handheld device andpermitting passage of optical signals having a wavelength correspondingto bacterial fluorescence of the illuminated portion of the target;collecting bacterial fluorescence data regarding the illuminated portionof the target with an image sensor of the handheld device, wherein thecollected bacterial fluorescence data comprises at least one imagecontaining data relating to bacterial autofluorescence responsive toillumination of the target and/or exogenous fluorescence of bacteriaresponsive to illumination of the target; identifying pixels in theimage having an intensity above a predetermined threshold; anddetermining a bacterial load of the illuminated portion of the target bysumming the identified pixels, wherein the bacterial load is themeasurable bacteria in the target.
 2. The method of claim 1, whereindirectly illuminating at least a portion of a target with a uniformfield of excitation light includes illuminating the target with at leastone light source configured to emit excitation light having a wavelengthof about 405 nm ±10 nm.
 3. The method of claim 1, wherein directlyilluminating at least a portion of a target with a uniform field ofexcitation light includes illuminating the target with excitation lightfrom first and second light sources.
 4. The method of claim 3, whereinilluminating the target with excitation light from first and secondlight sources includes simultaneously illuminating the target with firstand second light sources configured to emit excitation light having awavelength of about 405 nm ±10 nm.
 5. The method of claim 1, whereincollecting bacterial fluorescence data with an image sensor of thehandheld device includes detecting the filtered signals with an imagesensor of a digital camera contained within the housing.
 6. The methodof claim 5, wherein the digital camera is embedded in a wirelesscommunication device.
 7. The method of claim 1, wherein the target isone or more of a wound, normal skin, a chronic wound, a burn wound, anon-healing wound, and an ulcer.
 8. The method of claim 7, whereinilluminating at least a portion of a target comprises illuminating aperiphery of a wound.
 9. The method of claim 7, further comprisingtracking the bacterial load of the target over time.
 10. The method ofclaim 9, wherein the target is a wound and wherein changes in thebacterial load of the wound are indicative of wound healing or woundinfection.
 11. The method of claim 1, wherein illuminating at least aportion of a target comprises illuminating a wound prior to cleaningand/or debridement, and wherein determining a bacterial load of theilluminated portion of the target comprises determining the bacterialload of the wound prior to cleaning and/or debridement.
 12. The methodof claim 11, further comprising illuminating the portion of the woundsubsequent to cleaning and/or debridement and determining the bacterialload of the wound subsequent to cleaning and/or debridement.
 13. Themethod of claim 12, further comprising determining an efficacy of thecleaning and/or debridement of the wound based on the bacterial load ofthe wound subsequent to the cleaning and/or debridement.
 14. The methodof claim 1, further comprising determining a treatment strategy for thetarget based at least in part on the bacterial load.
 15. The method ofclaim 1, further comprising displaying an image indicative of thebacterial load of the target on a display of the handheld device. 16.The method of claim 15, wherein displaying the image indicative of thebacterial load of the target includes co-registering bacterial load datarelative to at least one of target topography, target anatomy, targetarea, target depth, target volume, target margins, and necrotic tissuein the image.
 17. The method of claim 1, further comprising convertingthe summed pixels into an area measurement to provide an area of thetarget containing bacteria.
 18. The method of claim 1, furthercomprising: illuminating the target with white light; detecting signalsresponsive to white-light illumination of the target with the imagesensor of the handheld device; determining a number of pixelsrepresentative of the target; and determining an area of the targetbased on the number of pixels.
 19. The method of claim 1, wherein the atleast one image includes at least one RGB image and further comprising:separating the at least one RGB image into individual channels; andconverting individual green and red image channels from the at least oneRGB image to gray scale, wherein identifying pixels having an intensityabove a predetermined threshold comprises identifying pixels whose grayscale intensity is above the predetermined threshold.
 20. A system foracquiring data regarding a wound in tissue, comprising: at least oneexcitation light source configured to directly illuminate a target witha uniform field of excitation light and cause bacteria present on and/orin the target to fluoresce; an optical sensor configured to detectsignals responsive to illumination of bacteria on and/or in the targetwith excitation light, each signal indicative of at least one ofendogenous fluorescence, exogenous fluorescence, absorbance, andreflectance in the illuminated target; a processor configured to receivethe detected signals, to identify pixels corresponding to bacterialfluorescence emitted from the target and having an intensity above apredetermined threshold, to calculate bacterial load of the target basedon the identified pixels, and to output data regarding the bacterialload of the target; and a display for displaying the output dataregarding the illuminated target output by the processor.
 21. The systemof claim 20, further comprising a filter configured to permit passage ofoptical signals responsive to illumination of the bacteria on and/or inthe target with the excitation light and having a wavelengthcorresponding to bacterial autofluorescence and exogenous fluorescenceof bacteria.
 22. The system of claim 21, wherein the filter isconfigured to permit optical signals having a wavelength greater thanabout 600 nm to pass through the filter to the optical sensor.
 23. Thesystem of claim 20, wherein the at least one excitation light source isconfigured to emit excitation light having a wavelength of about 405 nm±10 nm.
 24. The system of claim 20, further comprising a white-lightsource being positioned to illuminate the target with white light duringwhite-light imaging.
 25. The system of claim 24, wherein the processoris further configured to determine a size of the target based on anumber of pixels associated with the target identified in a white-lightimage of the target.
 26. The system of claim 20, wherein the processoris further configured to identify at least one of a target cleaningprotocol, a target debridement protocol, a target sampling protocol, atarget treatment protocol, and other target intervention strategy basedat least in part on the bacterial load.
 27. The system of claim 20,wherein the processor is further configured to identify an indication ofat least one of wound infection, wound healing, and a wound healingfailure based at least in part on the bacterial load.
 28. The system ofclaim 20, wherein the processor is further configured to track changesin the bacterial load of the target over time.
 29. The system of claim28, wherein the processor is further configured to recommend adjustmentsto an intervention strategy based, at least in part, on changes in thebacterial load of the illuminated portion of the target over time. 30.The system of claim 20, wherein the processor is further configured toreceive signals corresponding to tissue fluorescence and identify tissuecomponents of the target.
 31. The system of claim 30, wherein theprocessor is further configured to track changes in the tissuecomponents of the target over time.
 32. The system of claim 31, whereinthe processor is further configured to recommend adjustments to anintervention strategy based, at least in part, on changes in the tissuecomponents of the illuminated portion of the target over time.
 33. Amethod of obtaining diagnostic data regarding a target, comprising:directly illuminating at least a portion of a target with a uniformfield of excitation light emitted by at least one excitation lightsource connected to a housing of a handheld device, the housingincluding an enclosure for receiving a wireless communication devicehaving a digital camera, the at least one light source emitting at leastone wavelength or wavelength band causing bacteria in and/or on theilluminated portion of the target to fluoresce; capturing fluorescenceof the bacteria responsive to illumination of the illuminated portion ofthe target with an image sensor of the digital camera of the wirelesscommunication device, the wireless communication device being secured inthe housing; identifying pixels in the captured fluorescence having anintensity above a predetermined threshold; determining a bacterial loadof the illuminated portion of the target based on the number of pixelsidentified; and tracking changes in bacterial load of the target overtime.
 34. The method of claim 33, further comprising determining anintervention strategy based at least in part on the bacterial load ofthe illuminated portion of the target.
 35. The method of claim 34,further comprising adjusting the intervention strategy based on changesin the bacterial load of the illuminated portion of the target overtime.
 36. The method of claim 33, further comprising: illuminating thetarget with white light; detecting signals responsive to white-lightillumination of the target with the image sensor of the digital cameraof the wireless communication device; determining a number of pixelsrepresentative of the target; and determining an area of the targetbased on the number of pixels.
 37. The method of claim 36, furthercomprising tracking changes in the area of the target over time.
 38. Themethod of claim 37, further comprising determining an interventionstrategy based at least in part on the area of the target.
 39. Themethod of claim 38, further comprising adjusting the interventionstrategy based on changes in the area of the target overtime.